CA2679426A1

CA2679426A1 - Inhibitors of serine proteases

Info

Publication number: CA2679426A1
Application number: CA002679426A
Authority: CA
Inventors: Luc Farmer; Randy Scott Bethiel; Dylan Jacobs; Robert B. Perni; John Maxwell; Kevin Cottrell; Summer Halas
Original assignee: Individual
Current assignee: Vertex Pharmaceuticals Inc
Priority date: 2007-02-27
Filing date: 2008-02-20
Publication date: 2008-09-04
Also published as: EP2134717A2; WO2008106058A3; MX2009009176A; JP2010519329A; AU2008219704A1; US20100272681A1; NZ579295A; US8575208B2; CN101903392A; WO2008106058A2

Abstract

The present invention relates to compounds that inhibit serine protease activity, particularly the activity of hepatitis C virus NS3-NS4A protease. As such, they act by interfering with the life cycle of the hepatitis C virus and are also useful as antiviral agents. The invention further relates to compositions comprising these compounds either for ex vivo use or for administration to a patient suffering from HCV infection. The invention also relates to methods of treating an HCV infection in a patient by administering a composition comprising a compound of this invention.

Description

INHIBITORS OF SERINE PROTEASES

CLAIM OF PRIORTY

[0001] This application claims priority to U.S. provisional application serial number 60/903,814, filed February 27, 2007, which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION

[0002] The present invention relates to compounds that inhibit serine protease activity, particularly the activity of hepatitis C virus NS3-NS4A protease. As such, they act by interfering with the life cycle of the hepatitis C virus and are also useful as antiviral agents.
The invention further relates to compositions comprising these compounds either for ex vivo use or for administration to a patient suffering from HCV infection. The invention also relates to methods of treating an HCV infection in a patient by administering a composition comprising a compound of this invention.
BACKGROUND OF THE INVENTION

[0003] Infection by hepatitis C virus ("HCV") is a compelling human medical problem.
HCV is recognized as the causative agent for most cases of non-A, non-B
hepatitis, with an estimated human sero-prevalence of 3% globally [A. Alberti et al., "Natural History of Hepatitis C," J. Hepatology, 31., (Suppl. 1), pp. 17-24 (1999)]. Nearly four million individuals may be infected in the United States alone [M.J. Alter et al., "The Epidemiology of Vira1 Hepatitis-in-the United States"; Gastroenterol: Clin. North Am., 23, pp. 437-455 (1994); M. J. Alter "Hepatitis C Virus Infection in the United States," J.
Hepatology, 31., (Suppl. 1), pp. 88-91 (1999)].

[0004] Upon first exposure to HCV only about 20% of infected individuals develop acute clinical hepatitis while others appear to resolve the infection spontaneously.
In almost 70%
of instances; however, the virus establishes a chronic infection that persists for decades [S.
Iwarson, "The Natural Course of Chronic Hepatitis," FEMS Microbiology Reviews, 14, pp.
201-204 (1994); D. Lavanchy, "Global Surveillance and Control of Hepatitis C,"
J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usually results in recurrent and progressively worsening liver inflammation, which often leads to more severe disease states such as cirrhosis and hepatocellular carcinoma [M.C. Kew, "Hepatitis C and Hepatocellular Carcinoma", FEMS
Microbiologr Reviews, 14,-pp. 211-220 (1994); I. Saito et. al., "Hepatitis C
Virus Infection is Associated with the Development of Hepatocellular Carcinoma," Proc. Natl.
Acad. Sci. USA, 87, pp. 6547-6549 (1990)]. Unfortunately, there are no broadly effective treatments for the debilitating progression of chronic HCV.

[0005] The HCV genome encodes a polyprotein of 3010-3033 amino acids [Q.L.
Choo, et.
al., "Genetic Organization and Diversity of the Hepatitis C Virus." Proc.
Natl. Acad. Sci.
USA, 88, pp. 2451-2455 (1991); N. Kato et al., "Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis," Proc.
Natl. Acad.
Sci. USA, 87, pp. 9524-9528 (1990); A. Takamizawa et. al., "Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers," J. Virol., 65, pp.

(1991)]. The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication. The NS proteins are derived by proteolytic cleavage of the polyprotein [R. Bartenschlager et. al., "Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions,"
J. Virol., 67, pp. 3835-3844 (1993); A. Grakoui et. al., "Characterization of the Hepatitis C
Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites," J. Virol., 67, pp. 2832-2843 (1993); A. Grakoui et. al., "Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products," J. Virol., 67, pp. 1385-1395 (1993); L. Tomei et. al., "NS3 is a serine protease required for processing of hepatitis C
virus polyprotein", J. Virol., 67, pp. 4017-4026 (1993)].

[0006] The HCV NS protein 3 (NS3) contains a serine protease activity that helps process the majority of the viral enzymes, and is thus considered essential for viral replication and infectivity. It is known that mutations in the yellow fever virus NS3 protease decrease viral infectivity [Chambers, T.J. et. al., "Evidence that the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein", Proc. Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)].
The first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein [C. Lin et al., "Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics", J. Virol., 68, pp. 8147-8157 (1994)].

[0007] The HCV NS3 serine protease and its associated cofactor, NS4A, helps process all of the viral enzymes, and is thus considered essential for viral replication.
This processing appears to be analogous to that carried out by the human immunodeficiency virus aspartyl protease, which is also involved in viral enzyme processing. HIV protease inhibitors, which inhibit viral protein processing, are potent antiviral agents in man indicating that interrupting this stage of the viral life cycle results in therapeutically active agents.
Consequently HCV
NS3 serine protease is also an attractive target for drug discovery.

[0008] There are not currently any satisfactory anti-HCV agents or treatments.
Until recently, the only established therapy for HCV disease was interferon treatment. However, interferons have significant side effects [M. A. Walker et al., "Hepatitis C
Virus: An Overview of Current Approaches and Progress," DDT, 4, pp. 518-29 (1999); D.
Moradpour et al., "Current and Evolving Therapies for Hepatitis C," Eur. J.
Gastroenterol. Hepatol., 11, pp. 1199-1202 (1999); H. L. A. Janssen et al. "Suicide Associated with Alfa-Interferon Therapy for Chronic Viral Hepatitis," J. Hepatol., 21, pp. 241-243 (1994);
P.F. Renault et al., "Side Effects of Alpha Interferon," Seminars in Liver Disease, 9, pp. 273-277.
(1989)] and induce long term remission in orily a fraction (z 25 %) of cases [0. Weiland, "Interferon Therapy in Chronic Hepatitis C Virus Infection", FEMS Microbiol. Rev., 14, pp.

(1994)]. Recent introductions of the pegylated forms of interferon (PEG-INTRONO and PEGASYSO) and the combination therapy of ribavirin and pegylated interferon (REBETROLO) have resulted in only modest improvements in remission rates and only partial reductions in side effects. Moreover, the prospects for effective anti-HCV vaccines remain uncertain.

[0009] Thus, there is a need for more effective anti-HCV therapies. Such inhibitors would have therapeutic potential as protease inhibitors, particularly as serine protease inhibitors, and more particularly as HCV NS3 protease inhibitors. Specifically, such compounds may be useful as antiviral agents, particularly as anti-HCV agents.
SUMMARY OF THE INVENTION

[0010] This invention relates to compounds of formula I

,~N Rs A
N

O
I
or a pharmaceutically acceptable salt thereof.
[0010] Ri is -ZARg, wherein each ZA is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZA are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRA-, -C(O)NRANRA-, -C(O)O-, -NRAC(O)O-, -0-, -NRAC(O)NRA-, -NRANRA-, -S-, -SO-, -SO2-, -NRA-, -SO2NRA-, or -NRASOzNR'4- provided that -0-, -NRANRA-, -NRAC(O)NRA-, or -NRASO2NRA- is not directly bound to the nitrogen ring atom of formula I. Each R8 is independently RA, halo, -OH, -CN, -NOz, -NH2, or -OCF3. Each RA is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
100111 R2 and R3 are -ZBR9, wherein each ZB is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of ZB are optionally and independently replaced by -C(O)-, =C(S)-, -C(O)NRB-, =C(O)NRBNRB-, -C(O)O-, -NRBC(O)O-, -NRBC(O)NRB-, -NRBNRB-, -S-, -SO-, -SO2-, -NRB-, -SOZNRB-, or -NRBSO2NRB-. Each R9 is independently RB, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RB is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0012] Alternatively, R2 and R3, together form an oxo group.
[0013] A is -0-, or -CR6R7-, wherein each R6 and R7 is -ZCRio, wherein each Zc is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of Zc are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRc-, -C(O)NRcNRc-, -C(O)O-, -NRcC(O)O-, -NRcC(O)NRc-, -NRcNRc-, -S-, -SO-, -SOZ-, -NRC-, -SOZNRc-, or -NRCSO2NRc-. Each Rio is independently Rc, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each Rc is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0014] R4 is -Z Ri 1, wherein each ZD is independently a bond or an optionally substituted branched or straight C i_1Z aliphatic chain wherein up to three carbon units of Z D are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR -, -C(O)NR NR -, -C(O)O-, -NR C(O)O-, -0-, -NR C(O)NR -, -NR NR -, -S-, -SO-, -SO2-, -NR -, -S02NR -, or -NR SO2NR -, provided that -SO-, -SO2-, or -S02NR - is not directly bound to the carbonyl adjacent to R4. Each Ri I is independently R , halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each R is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0015] R5 is -ZER12, wherein each ZE is independently a bond or an optionally substituted branched or straight Ci_12 aliphatic chain wherein up to three carbon units of ZD are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRE-, -C(O)NRENRE-, -C(O)O-, -NREC(O)O-, -0-, -NREC(O)NRE-, -NRENRE-, -S-, -SO-, -SO2-, -NRE-, -SO2NRE-, or -NRESO2NRE-. Each R12 is independently RE, halo, -OH, -CN, -NOz, -NH2, or -OCF3. Each RE is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0016] In some aspects, the invention features a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof in an amount effective to inhibit a serine protease; and an acceptable carrier, adjuvant or vehicle.
The composition may include an additional agent selected from an immunomodulatory agent; an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; a cytochrome P-450 inhibitor; or combinations thereof. The immunomodulatory agent is a-, (3-, or y-interferon or thymosin; said antiviral agent is ribavirin, amantadine, or telbivudine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV
helicase, polymerase, or metalloprotease. Cytochrome P-450 inhibitor may be ritonavir.
[0017] In other aspects, a method of inhibiting the activity of a serine protease comprising the step of contacting said serine protease with a compound of formula I. The serine protease may be an HCV NS3 protease. The methods also include treating an HCV infection in a patient by administering a compound of formula I. The method may also include administering to said patient an additional agent selected from an immunomodulatory agent;
an antiviral agent; a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; or combinations thereof; wherein said additional agent is administered to said patient in the same dosage form as the serine protease inhibitor or as a separate dosage form. The immunomodulatory agent is a-, (3-, or y-interferon or thymosin; said antiviral agent is ribavarin or amantadine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.
[0018] In still other aspects, a method of eliminating or reducing HCV
contamination of a biological sample or medical or laboratory equipment, includes the step of contacting said biological sample or medical or laboratory equipment with a compound of formula I. The sample or equipment may be selected from blood, other body fluids, biological tissue, a surgical instrument, a surgical garment, a laboratory instrument, a laboratory garment, a blood or other body fluid collection apparatus; a blood or other body fluid storage material.
{0019] The. compounds of the invention, as described herein, also exhibit advantageous PK
properties and/or increased potency.
[00201 The invention also relates to compositions that comprise the above compounds and the use thereof; methods of preparing compounds of formula I, and methods of assaying compounds for serine protease activity. Such compositions may be used to pre-treat devices that are to be inserted into a patient, to treat biological samples, or for direct administration to a patient. In each case, the composition will be used to lessen the risk of or the severity of the HCV infection.
[0021] In other aspects, the invention features certain compounds as described generically and specifically below. Such specific descriptions are illustrative only and are not meant to limit scope of the compounds or uses thereof.
'DETAILED DESCRIPTION
1. DEFINITIONS
[0022] For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
[0023] As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention.
[0024] As used herein the term "aliphatic" encompasses the terms alkyl, alkenyl, alkynyl, each of which being optionally substituted as set forth below.
[0025] As used herein, an "alkyl" group refers to a saturated aliphatic hydrocarbon group containing 1-8 (e.g., 1-12, 1-6, or 1-4) carbon atoms. An alkyl group can be straight or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or 2-ethylhexyl. An alkyl group can be substituted (i.e., optionally substituted) with one or more substituents such as halo, phospho, cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic [e.g., heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro, cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g., aliphaticamino, cycloaliphaticamino, or heterocycloaliphaticamino], sulfonyl [e.g., aliphatic-S02-], sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy.
Without limitation, some examples of substituted alkyls include carboxyalkyl (such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl), cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as (alkyl-S02-amino)alkyl), aminoalkyl, amidoalkyl, (cycloaliphatic)alkyl, or haloalkyl.
100261 As used herein, an "alkenyl" group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-12, 2-6, or 2-4) carbon atoms and at least one double bond. Like an alkyl group, an alkenyl group can be straight or branched. Examples of an alkenyl group include, but are not limited to allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can be optionally substituted with one or more substituents such as halo, phospho, cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic [e.g., heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl, alkoxy, aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl, (cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro, cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g., aliphaticamino, cycloaliphaticamino, heterocycloaliphaticamino, or aliphaticsulfonylamino], sulfonyl [e.g., alkyl-S02-, cycloaliphatic-SO2-, or aryl-S02-], sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy.
Without limitation, some examples of substituted alkenyls include cyanoalkenyl, alkoxyalkenyl, acylalkenyl, hydroxyalkenyl, aralkenyl, (alkoxyaryl)alkenyl, (sulfonylamino)alkenyl (such as (alkyl-S02-amino)alkenyl), aminoalkenyl, amidoalkenyl, (cycloaliphatic)alkenyl, or haloalkenyl.
[0027] As used herein, an "alkynyl" group refers to an aliphatic carbon group that contains 2-8 (e.g., 2-12, 2-6, or 2-4) carbon atoms and has at least one triple bond.
An alkynyl group can be straight or branched. Examples of an alkynyl group include, but are not limited to, propargyl and butynyl. An alkynyl group can be optionally substituted with one or more substituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy, cyano, halo, hydroxy, sulfo, mercapto, sulfanyl [e.g., aliphaticsulfanyl or cycloaliphaticsulfanyl], sulfinyl [e.g., aliphaticsulfinyl or cycloaliphaticsulfinyl], sulfonyl [e.g., aliphatic-S02-, aliphaticamino-S02-, or cycloaliphatic-S02-], amido [e.g., aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino, heteroarylcarbonylamino or heteroarylaminocarbonyl], urea, thiourea, sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, acyl [e.g., (cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl], amino [e.g., aliphaticamino], sulfoxy, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.
100281 As used herein, an "amido" encompasses both "aminocarbonyl" and "carbonylamino". These terms when used alone or in connection with another group refer to an amido group such as -N(Rx)-C(O)-RY or -C(O)-N(Rx)Z, when used terminally, and -C(O)-N(Rx)- or -N(Rx)-C(O)- when used internally, wherein Rx and RY are, defined below.
Examples of amido groups include alkylamido (such as alkylcarbonylamino or alkylaminocarbonyl), (heterocycloaliphatic)amido, (heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyl)alkylamido, arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.
[0029] As used herein, an "amino" group refers to -NRxRY wherein each of Rx and RY is independently hydrogen, aliphatic, cycloaliphatic, (cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl, ((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or (heteroaraliphatic)carbonyl, each of which being defined herein and being optionally substituted. Examples of amino groups include alkylamino, dialkylamino, or arylamino.
When the term "amino" is not the terminal group (e.g., alkylcarbonylamino), it is represented by -NRx-. Rx has the same meaning as defined above.
[0030] As used herein, an "aryl" group used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl" refers to monocyclic (e.g., phenyl);
bicyclic (e.g., indenyl, naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyl tetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systems in which the monocyclic ring system is aromatic or at least one of the rings in a bicyclic or tricyclic ring system is aromatic. The bicyclic and tricyclic groups include benzofused 2-3 membered carbocyclic rings. For example, a benzofused group includes phenyl fused with two or more C4_8 carbocyclic moieties. An aryl is optionally substituted with one or more substituents including aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic;
(cycloaliphatic)aliphatic;
heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl;
alkoxy;
(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;
(araliphatic)oxy;
(heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of a benzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl [ e.g., aliphaticcarbonyl;
(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;
(araliphatic)carbonyl;
(heterocycloaliphatic)carbonyl; ((heterocycloaliphatic)aliphatic)carbonyl; or (heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphatic-S02- or amino-SO2-];
sulfinyl [e.g., aliphatic-S(O)- or cycloaliphatic-S(O)-]; sulfanyl [e.g., aliphatic-S-];
cyano; halo; hydroxy;
mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl.
Alternatively, an aryl can be unsubstituted.
[0031] Non-limiting examples of substituted aryls include haloaryl [e.g., mono-, di ( such as p,m-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl [e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and (alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl, (((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl, (arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl]; aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl]; (cyanoalkyl)aryl;
(alkoxy)aryl;
(sulfamoyl)aryl [e.g., (aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;
(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl, ((carboxy)alkyl)aryl;
(((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl;
(((alkylsulfonyl)amino)alkyl)aryl;
((heterocycloaliphatic)carbonyl)aryl; ((alkylsulfonyl)alkyl)aryl;
(cyanoalkyl)aryl;
(hydroxyalkyl)aryl; (alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl; p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl; or (m-(heterocycloaliphatic)-o-(alkyl))aryl.
[0032] As used herein, an "araliphatic" such as an "aralkyl" group refers to an aliphatic group (e.g., a C1_4 alkyl group) that is substituted with an aryl group.
"Aliphatic," "alkyl,"
and "aryl" are defined herein. An example of an araliphatic such as an aralkyl group is benzyl.
[0033] As used herein, an "aralkyl" group refers to an alkyl group (e.g., a C1_4 alkyl group) that is substituted with an aryl group. Both "alkyl" and "aryl" have been defined above. An example of an aralkyl group is benzyl. An aralkyl is optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl, including carboxyalkyl, hydroxyalkyl, or haloalkyl such as trifluoromethyl], cycloaliphatic [e.g., cycloalkyl or cycloalkenyl], (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amido [e.g., aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, -(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, or heteroaralkylcarbonylamino], cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0034] As used herein, a "bicyclic ring system" includes 8-12 (e.g., 9, 10, or 11) membered structures that form two rings, wherein the two rings have at least one atom in common (e.g., 2 atoms in common). Bicyclic ring systems include bicycloaliphatics (e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclic heteroaryls.
[0035] As used herein, a "cycloaliphatic" group encompasses a "cycloalkyl"
group and a "cycloalkenyl" group, each of which being optionally substituted as set forth below.
As used herein, a "cycloalkyl" group refers to a saturated carbocyclic mono-or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbomyl, cubyl, octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1 ]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl, or ((aminocarbonyl)cycloalkyl)cycloalkyl. A "cycloalkenyl" group, as used herein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or more double bonds. Examples of cycloalkenyl groups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl, bicyclo[2.2.2]octenyl, or bicyclo[3.3.1]nonenyl. A cycloalkyl or cycloalkenyl group can be optionally substituted with one or more substituents such as phosphor, aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic) aliphatic, heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino, ((heterocycloaliphatic)aliphatic)carbonylamino, (heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro, carboxy [e.g., HOOC-, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl], cyano, halo, hydroxy, mercapto, sulfonyl [e.g., alkyl-S02- and aryl-SOz-], sulfinyl [e.g., alkyl-S(O)-], sulfanyl [e.g., alkyl-S-], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0036] As used herein, the term "heterocycloaliphatic" encompasses a heterocycloalkyl group and a heterocycloalkenyl group, each of which being optionally substituted as set forth below.
[0037] As used herein, a "heterocycloalkyl" group refers to a 3-10 membered mono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- or bicyclic) saturated ring structure, in which one or more of the ring atoms is a heteroatom (e.g., N, 0, S, or combinations thereof). Examples of a heterocycloalkyl group include piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl, octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl, octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl, octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl, 1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1 ]octyl, and 2,6-dioxa-tricyclo[3.3.1.03'7 ]nonyl. A monocyclic heterocycloalkyl group can be fused with a phenyl moiety to form structures, such as tetrahydroisoquinoline, which would be categorized as heteroaryls. A "heterocycloalkenyl" group, as used herein, refers to a mono- or bicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ring structure having one or more double bonds, and wherein one or more of the ring atoms is a heteroatom (e.g., N, 0, or S). Monocyclic and bicyclic heterocycloaliphatics are numbered according to standard chemical nomenclature.
[0038] A heterocycloalkyl or heterocycloalkenyl group can be optionally substituted with one or more substituents such as phosphor, aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic)aliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic, aryl, heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl, heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino, (cycloaliphatic)carbonylamino, ((cycloaliphatic) aliphatic)carbonylamino, (aryl)carbonylamino, (araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino, ((heterocycloaliphatic) aliphatic)carbonylamino, (heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro, carboxy [e.g., HOOC-, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl, (heterocycloaliphatic)carbonyl, ((heterocycloaliphatic)aliphatic)carbonyl, or (heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto, sulfonyl [e.g., alkylsulfonyl or arylsulfonyl], sulfinyl [e.g., alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0039] A"heteroaryl" group, as used herein, refers to a monocyclic, bicyclic, or tricyclic ring system having 4 to 15 ring atoms wherein one or more of the ring atoms is a heteroatom (e.g., N, 0, S, or combinations thereof) and in which the monocyclic ring system is aromatic or at least one of the rings in the bicyclic or tricyclic ring systems is aromatic. A heteroaryl group includes a benzofused ring system having 2 to 3 rings. For example, a benzofused group includes benzo fused with one or two 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples of heteroaryl are azetidinyl, pyridyl, 1 H-indazolyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine, dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl,cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.
100401 Without limitation, monocyclic heteroaryls include furyl, thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl,.thazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.
Monocyclic heteroaryls are numbered according to standard chemical nomenclature.
[0041] Without limitation, bicyclic heteroaryls include indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl, benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl. Bicyclic heteroaryls are numbered according to standard chemical nomenclature.
[0042] A heteroaryl is optionally substituted with one or more substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;
heterocycloaliphatic;. (heterocycloaliphatic)aliphatic; aryl; heteroaryl;
alkoxy;
(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;
(araliphatic)oxy;
(heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic or tricyclic heteroaryl); carboxy; amido; acyl [ e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;
((cycloaliphatic)aliphatic)carbonyl;
(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;
((heterocycloaliphatic)aliphatic)carbonyl; or (heteroaraliphatic)carbonyl];
sulfonyl [e.g., aliphaticsulfonyl or aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl];
sulfanyl [e.g., aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy; urea;
thiourea; sulfamoyl;
sulfamide; or carbamoyl. Alternatively, a heteroaryl can be unsubstituted.
[0043] ' Non-limiting examples of substituted heteroaryls include (halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl]; (carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl];
cyanoheteroaryl; aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g., aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl, ((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl, (((heteroaryl)amino)carbonyl)heteroaryl, ((heterocycloaliphatic)carbonyl)heteroaryl, and ((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl; (alkoxy)heteroaryl;
(sulfamoyl)heteroaryl [e.g., (aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g., (alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl; (alkoxyalkyl)heteroaryl;
(hydroxy)heteroaryl; ((carboxy)alkyl)heteroaryl;
(((dialkyl)amino)alkyl]heteroaryl;
(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;
(nitroalkyl)heteroaryl;
(((alkylsulfonyl)amino)alkyl)heteroaryl; ((alkylsulfonyl)alkyl)heteroaryl;
(cyanoalkyl)heteroaryl; (acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl];
(alkyl)heteroaryl, and (haloalkyl)heteroar.yl [e.g., trihaloalkylheteroaryl].
[0044] A "heteroaraliphatic (such as a heteroaralkyl group) as used herein, refers to an aliphatic group (e.g., a C1 -4 alkyl group) that is substituted with a heteroaryl group.
"Aliphatic," "alkyl," and "heteroaryl" have been defined above.
[0045] A "heteroaralkyl" group, as used herein, refers to an alkyl group (e.g., a C i-4 alkyl group) that is substituted with a heteroaryl group. Both "alkyl" and "heteroaryl" have been defined above. A heteroaralkyl is optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0046] As used herein, "cyclic moiety" and "cyclic group" refer to mono-, bi-, and tri-cyclic ring systems including cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl, each of which has been previously defined.
[0047] As used herein, a "bridged bicyclic ring system" refers to a bicyclic heterocyclicalipahtic ring system or bicyclic cycloaliphatic ring system in which the rings are bridged. Examples of bridged bicyclic ring systems include, but are not limited to, adamantanyl, norbornanyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.2.3]nonyl, 2-oxabicyclo[2.2.2]octyl, 1-azabicyclo[2.2.2]octyl, 3-azabicyclo[3.2.1]octyl, and 2,6-dioxa-tricyclo[3.3.1.03'7]nonyl. A bridged bicyclic ring system can be optionally substituted with one or more substituents such as alkyl (including carboxyalkyl, hydroxyalkyl, and haloalkyl such as trifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl, alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino, cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino, (heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo, hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0048] As used herein, an "acyl" group refers to a formyl group or Rx-C(O)-(such as alkyl-C(O)-, also referred to as "alkylcarbonyl") where Rx and "alkyl" have been defined previously. Acetyl and pivaloyl are examples of acyl groups.
[0049] As used herein, an "aroyl" or "heteroaroyl" refers to an aryl-C(O)- or a heteroaryl-C(O)-. The aryl and heteroaryl portion of the aroyl or heteroaroyl is optionally substituted as previously defined.
[0050] As used herein, an "alkoxy" group refers to an alkyl-O- group where "alkyl" has been defined previously.
[0051] As used herein, a "carbamoyl" group refers to a group having the structure -O-CO-NRXRY or--NRx-CO-O-RZ, wherein Rx and RY have been defined above and RZ
can be aliphatic, aryl, araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

[0052] As used herein, a "carboxy" group refers to -COOH, -COORx, -OC(O)H, -OC(O)Rx, when used as a terminal group; or -OC(O)- or -C(O)O- when used as an internal group.
[0053] As used herein, a "haloaliphatic" group refers to an aliphatic group substituted with 1-3 halogen. For instance, the term haloalkyl includes the group -CF3.
[0054] As used herein, a "mercapto" group refers to -SH.
[0055] As used herein, a "sulfo" group refers to -SO3H or -SO3Rx when used terminally or -S(O)3- when used internally.
[0056] As used herein, a "sulfamide" group refers to the structure -NRx-S(O)2-NRYRZ when used terminally and -NRX-S(O)2-NRY- when used internally, wherein Rx, RY, and Rz have been defined above.
[0057] As used herein, a "sulfonamide" group refers to the structure -S(O)Z-NRxRY or -NRx-S(O)2-RZ when used terminally; or -S(O)Z-NRx- or -NRX -S(O)2- when used internally, wherein Rx, RY, and Rz are defined above.
[0058] As used herein a "sulfanyl" group refers to -S-RX when used terminally and -S-when used internally, wherein Rx has been defined above. Examples of sulfanyls include aliphatic-S-, cycloaliphatic-S-, aryl-S-, or the like.
[0059] As used herein a "sulfinyl" group refers to -S(O)-Rx when used terminally and -S(O)- when used internally, wherein Rx has been defined above. Exemplary sulfinyl groups include-aliphatic-S(O)-, aryl-S(O)-,-(cycloaliphatic(aliphatic))-S(O)-, cycloalkyl-S(O)-, heterocycloaliphatic-S(O)-, heteroaryl-S(O)-, or the like.
[0060] As used herein, a "sulfonyl" group refers to-S(O)2-Rx when used terminally and -S(O)2- when used internally, wherein RX has been defined above. Exemplary sulfonyl groups include aliphatic-S(O)Z-, aryl-S(O)Z-, (cycloaliphatic(aliphatic))-S(O)2-, cycloaliphatic-S(O)z-, heterocycloaliphatic-S(O)Z-, heteroaryl-S(O)2-, (cycloaliphatic(amido(aliphatic)))-S(O)Z-or the like.
[0061] As used herein, a "sulfoxy" group refers to -O-SO-Rx or -SO-O-Rx, when used terminally and -O-S(O)- or -S(O)-O- when used internally, where Rx has been defined above.
[0062] As used herein, a "halogen" or "halo" group refers to fluorine, chlorine, bromine or iodine.
40063] As used herein, an "alkoxycarbonyl," which is encompassed by the term carboxy, used alone or in connection with another group refers to a group such as alkyl-O-C(O)-.
[0064] As used herein, an "alkoxyalkyl" refers to an alkyl group such as alkyl-O-alkyl-, wherein alkyl has been defined above.

[0065] As used herein, a "carbonyl" refer to -C(O)-.
100661 As used herein, an "oxo" refers to =0.
100671 As used herein, the term "phospho" refers to phosphinates and phosphonates.
Examples of phosphinates and phosphonates include -P(O)(RP)2, wherein RP is aliphatic, alkoxy, aryloxy, heteroaryloxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy aryl, heteroaryl, cycloaliphatic or amino.
[0068] As used herein, an "aminoalkyl" refers to the structure (Rx)2N-alkyl-.
100691 As used herein, a "cyanoalkyl" refers to the structure (NC)-alkyl-.
[0070] As used herein, a "urea" group refers to the structure -NRX-CO-NRYRZ
and a "thiourea" group refers to the structure -NRx-CS-NRYRZ when used terminally and -NRX-CO-NRY- or -NRx-CS-NRY- when used internally, wherein Rx, RY, and Rz have been defined above.
[0071] As used herein, a "guanidine" group refers to the structure -N=C(N(RXRY))N(RXRY) or -NRx-C(=NRX)NRxRY wherein Rx and RY have been defined above.
[0072] As used herein, the term "amidino" group refers to the structure -C=(NRX)N(RxRY) wherein Rx and RY have been defined above.
[0073] In general, the term "vicinal" refers to the placement of substituents on a group that includes two or more carbon atoms, wherein the substituents are attached to adjacent carbon atoms.
[0074] In general, the term "geminal" refers to the-placement of substituents on a group that includes two or more carbon atoms, wherein the substituents are attached to the same carbon atom.
[0075] The terms "terminally" and "internally" refer to the location of a group within a substituent. A group is terminal when the group is present at the end of the substituent not further bonded to the rest of the chemical structure. Carboxyalkyl, i.e., Rx0(O)C-alkyl is an example of a carboxy group used terminally. A group is internal when the group is present in the middle of a substituent of the chemical structure. Alkylcarboxy (e.g., alkyl-C(0)0- or alkyl-OC(O)-) and alkylcarboxyaryl (e.g., alkyl-C(0)0-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy groups used internally.
[0076] As used herein, an "aliphatic chain" refers to a branched or straight aliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups). A straight aliphatic chain has the structure -[CH2],-, where v is 1-6. A branched aliphatic chain is a straight aliphatic chain that is substituted with one or more aliphatic groups. A branched aliphatic chain has the structure -[CHQ], where Q is hydrogen or an aliphatic group; however, Q shall be an aliphatic group in at least one instance. The term aliphatic chain includes alkyl chains, alkenyl chains, and alkynyl chains, where alkyl, alkenyl, and alkynyl are defined above.
[0077] The phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." As described herein, compounds of the invention can optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. As described herein, the variables RI, R2, R3, R4, R5, A and other variables contained in formulae described herein encompass specific groups, such as alkyl and aryl. Unless otherwise noted, each of the specific groups for the variables Ri, R2, R3, R4, R5, A, and other variables contained therein can be optionally substituted with one or more substituents described herein. Each substituent of a specific group is further optionally substituted with one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro, aryl, cycloaliphatic, heterocycloaliphatic, heteroaryl, haloalkyl, and alkyl. For instance, an alkyl group can be substituted with alkylsulfanyl and the alkylsulfanyl can be optionally substituted with one to three of halo, cyano, oxo, alkoxy, hydroxy, amino, nitro, aryl, haloalkyl, and alkyl. As an additional example, the cycloalkyl portion of a(cycloalkyl)carbonylamino can be optionally substituted with one to three of halo, cyano, alkoxy, hydroxy, nitro, haloalkyl, and alkyl. When two alkoxy groups are bound to the same atom or adjacent atoms, the two alkxoy groups can form a ring together with the atom(s) to which they are bound.
[0078] In general; the-term "substituted," whether preceded-by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. A ring substituent, such as a heterocycloalkyl, can be bound to another ring, such as a cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings share one common atom. As one of ordinary skill in the art will recognize, combinations of substituents envisioned by this invention are those combinations that result in the formation of stable or chemically feasible compounds.
[0079] The phrase "stable or chemically feasible," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40 C
or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
[0080] As used herein, an effective amount is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970). As used herein, "patient"
refers to a mammal, including a human.
[0081] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g.,, enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention.
Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.
II. COMPOUNDS
A. Generic Compounds [0082] In some aspects, the invention provides compounds of formula I useful for inhibiting serine protease activity and methods of inhibiting serine protease activity.
Compounds of formula I include:

RZ
Rj\ R3 A
N
Rs Ra O
or a pharmaceutically acceptable salt thereof.
[0083] R, is -ZAR8, wherein each ZA is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZA are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRA-, -C(O)NRANRA-, -C(O)O-, -NRAC(O)O-, -0-, -NRAC(O)NRA-, -NRANRA-, -S-, -SO-, -SO2-, -NR'4-, -SOZNRA-, or -NRASO2NRA- provided that -0-, -NRANRA-, -NRAC(O)NRA-, or -NRASO2NRA- is not directly bound to the nitrogen ring atom of formula I. Each R8 is independently RA, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RA is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0084] R2 and R3 are -ZBR9, wherein each ZB is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of ZB are optionally and independently replaced by =C(O)-, =C(S)-, -C(O)NRB-, -C(O)NRBNRB--C(O)O-, -NRBC(O)O-, -NRBC(O)NRB-, -NRBNRB-, -S-, -SO-, -SO2-, -NRB-, -SO2NRB-, or -NRBSOZNRB-. Each R9 is independently RB, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RB is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0085] Alternatively, R2 and R3, together form an oxo group.
[0086] A is -0-, or -CR6R7-, wherein each R6 and R7 is -Zc Rio, wherein each Zc is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of Zc are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRc-, -C(O)NRcNRc-, -C(O)O-, -NRcC(O)O-, -NRcC(O)NRc-, -NRcNRc-, -S-, -SO-, -SOZ-, -NRC-, -SO2NRc-, or -NRcSO2NRc-. Each Rio is independently Rc, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RC is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0087] R4 is -Z Ri i, wherein each Z is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of Z D are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR -, -C(O)NR NR -, -C(O)O-, -NR C(O)O-, -0-, -NR C(O)NR -, -NR NR -, -S-, -SO-, -SO2-, -NR -, -S02NR -, or -NR SOZNR -, provided that -SO-, -SO2-, or -S02NR - is not directly bound to the carbonyl adjacent to R4. Each R11 is independently R , halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each R D is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0088] R5 is -ZER12, wherein each ZE is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZE are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRE-, -C(O)NRENRE-, -C(O)O-, -NREC(O)O-, -0-, -NREC(O)NRE-, -NRENRE-, -S-, -SO-, -SO2-, -NRE-, -SO2NRE-, or -NRESO2NRE-. Each R12 is independently RE, halo, -OH, -CN, -NO2, -NH2, or -OCF3.
Each RE is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0089] Or, R4 and R5 together with the atoms to which they are attached form an optionally substituted heterocycloaliphatic ring.
B. Specific Compounds 1. Substituent Rl [0090] Ri is -ZAR8, wherein each ZA is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZA are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRA-, -C(O)NR'4NRA-, -C(O)O-, -NRAC(O)O-, -0-, -NRAC(O)NRA-, -NRANRA-, -S-, -SO-, -SO2-, -NRA-, -SO2NRA-, or -NRASO2NRA- provided that -0-, -NRANRA-, -NRAC(O)NRA-, or -NRASO2NRA- is not directly bound to the nitrogen ring atom of formula I_ Each R8 is independently RA, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RA is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0091] In several embodiments, Ri is an optionally substituted aliphatic. For example, R, is an alkyl, alkenyl, or alkynyl, each of which is optionally substituted with 1-4 substituents independently selected from halo, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic.
Alternatively, Ri is an optionally substituted alkyl. For example, Ri is methyl, ethyl, propyl, or butyl, each of which is optionally substituted with 1-4 substituents independently selected from halo, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic. In other examples, Ri is an unsubstituted alkyl. Alternatively, Ri is an optionally substituted aralkyl. In several examples, R, is an aryl-methyl, aryl-ethyl, or aryl-propyl, each of which is optionally substituted. Alternatively, Ri is phenylmethyl, phenylethyl, or phenylpropyl, each of which is optionally substituted with 1-3 substituents independently selected from halo, aryl, hydroxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl.
[0092] In several embodiments, R, is an optionally substituted aryl. For example, R, is a monocyclic or bicyclic aryl, each of which is optionally substituted. In other examples, Ri is phenyl optionally substituted with 1-3 substituents selected from halo, hydroxy, aliphatic, aryl, heteroaryl, cycloaliphatic, or heterocycloaliphatic.
[0093] In other examples, Ri is one selected from hydrogen, ethyl, phenyl, p-chloro-phenyl, and phenylmethyl.
2. Substituents R, and R3 [0094] R2 and R3 are -ZBR9, wherein each ZB is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of ZB are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRB-, -C(O)NRBNRB-, -C(O)O-, -NRBC(O)O-, -NRBC(O)NRB-, -NRBNRB-, -S-, -SO-, -SO2-, -NRB-, -SO2NRB-, or -NRBSO2NRB-. Each R9 is independently RB, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RB is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0095] Alternatively, R2 and R3, together form an oxo group.
[0096] In several embodiments, R2 and R3, together form an oxo group.
3. Group A
[0097] A is -0-, or -CR6R7-, wherein each R6 and R7 is -ZCRIo, wherein each Zc is independently a bond or an optionally substituted branched or straight C1_6 aliphatic chain wherein up to three carbon units of Zc are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRC-, -C(O)NRCNRc-, -C(O)O-, -NRcC(O)O-, -NRcC(O)NRc-, -NRcNRC-, -S-, -SO-, -SOZ-, -NRC-, -SO2NRc-, or -NRCSOZNRC-. Each Rio is independently RC, halo, -OH, -CN, -NOZ, -NH2, or -OCF3. Each Rc is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[0098] In several embodiments, A is -0-.

[0099] In other embodiments, A is -CR6R7-. For example, in some embodiments, A
is -CR6R7-, and one of R6 or R7 is hydrogen. In other examples, A is -CR6R7-, and one of R6 or R7 is hydrogen, and the other of R6 or R7 is optionally substituted C1_6 aliphatic. In other examples, A is -CR6R7-, and one of R6 or R7 is hydrogen, and the other of R6 or R7 is unsubstituted C1_6 aliphatic. Alternatively, A is -CR6R7-, and both of R6 or R7 are hydrogen.
4. Substituent R4 [00100] R4 is -Z R11, wherein each ZD is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of Z D are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR -, -C(O)NR NR -, -C(O)O-, -NR C(O)O-, -0-, -NR C(O)NR -, -NR NR -, -S-, -SO-, -S02-, -NR -, -S02NR -, or -NR SO2NR -, provided that -SO-, -SO2-, or -S02NR - is not directly bound to the carbonyl adjacent to R4. Each R, 1 is independently R , halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each R D is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[00101] In several embodiments, R4 is -Z R>>, wherein each Z is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZD are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR -, -C(O)NR NR -, -C(O)O-, -NR C(O)O-, -NR C(O)NR -, -NR NR -, -S-, -SO-, -SO2-, -NR -, -S02NR -, or -NR SOZNR -, provided that -SO-, -SO2-, or -S02NR - is not directly bound to the carbonyl of formula I. Each Rl l is independently R , halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each R D is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[00102] In still further embodiments, R4 is -Z1-V1-Z2-V2-Z3-V3 each of Vt, V2, and V3 is independently a bond, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when V1, V2, or V3 is the terminal group of R4; each of ZI, Z2, and Z3 is independently a bond, -C(O)-, -C(O)C(O)-, -C(S)-, -C(O)N(Q6)-, -N(Q6)C(O)-, -C(O)C(O)N(Q6)-, -0-,, SO-, -SO2-, -N(Q6)SO2-, -N(Q6)C(O)N(Q6)-, -N(Q6)C(S)N(Q6)-, -N(Q6)-, -N(Q6)SOZ-, -SO2N(Q6)-, -C(O)N(Q6)SO2-, -SO2N(Q6)C(O)-, or hydrogen when Zi, Z2, or Z3 is the terminal group of R4;
and each Q6 is independently hydrogen, or an optionally substituted aliphatic.

[00103] In other embodiments, R4 is an optionally substituted (aliphatic)amino wherein the aliphatic portion of R4 is -Z2-V2-Z3-V3 or -Z3-V3 wherein each of Z2 and Z3 is independently a bond, -C(O)-, -N(Q5)-, -CH(OH)-, -C(O)N(Q6)-, or -C(O)C(O)N(Q6)-; V2 is independently a bond, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic; and V3 is hydrogen, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic.
[00104] In still further embodiments, Z2 is -CH(OH)-, V2 is a bond, and Z3 is -C(O)N(Q6)-such that R4 is -N(Q6)-CH(OH)-C(O)-N(V3)(Q6).
[00105] In certain embodiments, R4 is an optionally substituted (aliphatic)amino, optionally substituted (cycloaliphatic)amino, an optionally substituted alkoxy, or hydroxy.
[00106] In still another embodiment, R4 is an alkoxy optionally substituted with 1-3 of halo, hydroxy, aliphatic, cycloaliphatic, or heterocycloaliphatic.
[00107] In several embodiments, R4 is amino. Examples of R4 include a mono-substituted amino. Additional examples of R4 include (cycloaliphatic(carbonyl(carbonyl(alkyl))))amino (amino(carbonyl(carbonyl(aliphatic))))amino, aliphatic(carbonyl(carbonyl(aliphatic))))amino, or (aryl(amino(carbonyl(carbonyl(aliphatic)))))amino, each of which is optionally substituted with 1 to 3 substituents.
[00108] In several embodiments, R4 is -NR4ZR'4Z, -SR4y, or -NR4Y-CR4xR'4x-Li-NR4Z-R4W, wherein R4Y is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R4w is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; each R4x and R'4X is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R4x and R'4x together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring; each L1 is -CH2-, -C(O)-, -CF2-, -C(O)C(O)-, -C(O)O-, -S(O)-, or -SO2-; each R4z or R'4Z is hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4z and R'4Z together with the nitrogen to which they are both attached may form an optionally substituted 3 to 7 membered heterocycloaliphatic ring.

[00109] In several embodiments, each R4x and R'4x is independently hydrogen, or optionally substituted aliphatic, optionally substituted cycloaliphatic, or optionally substituted (cycloaliphatic)aliphatic.
[00110] In several embodiments, Li is -C(O)C(O)- or -SO2-.
[00111] In several other embodiments, each R4w is hydrogen or optionally substituted cycloaliphatic.
[00112] In several embodiments, R4 is -NH-CHR4X-C(O)-C(O)-N(R4z)R4W.
[00113] In several embodiments, R4 is -NH-CHR4x-CH(OH)-C(O)-N(R4z)R4w.
1001141 In several embodiments, R4 is -NH-CHR4X-C(O)-C(O)-NHR4Z wherein -NHR4z is NH-cyclopropyl, -NH-Me, -NH-Et, -NH-iPr, -NH-nPr.
[00115] In several embodiments R4 is -NR4zR'4z, -SR4z wherein each R4z and R'4Z is independently hydrogen, alkyl, cycloalkyl or aralkyl. Non-limiting examples of R4z include methyl, ethyl, t-butyl, cyclopentyl, cyclohexyl and benzyl.
[00116] In other embodiments R4 is (-NH-CR4xR'4x- L1-C(O));-M; wherein each M
is independently -OH, -R4x, -NR4zR'4z, or -OR4x, each i is 1 or 2, and Li, R4z, R4x, and R'4Z are defined above.
[00117] In several embodiments R4 is (-NH-CR4zR'4z- LI-C(O));-M wherein L1 is -C(O)-, i is 1 and M is independently -R4x, -N(R4xR'4X), -OR4X, -NHSO2R4X, or -SR4x.
[00118] In some embodiments, R'4Z is H and R4z is aliphatic, (aryl)aliphatic or cycloaliphatic. Non-limiting examples of R4x include hydrogen, /

or ~ N
[00119] In some embodiments R'4X is H and R4x is optionally substituted aliphatic, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaliphatic or optionally substituted heteroaralkyl. Some non-limiting examples of R4x include:

IF .n F SH , nr 1 .n I rv, / C
lWtr ,,,,1lV`

~ I I
F F, or where c is 0-3.
[00120] In several embodiments, R4 is:

Rax N~Raw N
H 0 , wherein R4x is o r and R4W is or hydrogen.
[00121] In some embodiments, R4 is R56 Rax R'ax H
R NN, S-Rs7 H Ng~57 H 0 (O)m ~ (0)m or , wherein each R56 is independently optionally substituted C1_6 aliphatic; optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloaliphatic, or optionally substituted heterocycloaliphatic; each R57 is independently optionally substituted aliphatic, optionally substituted aryl, optionally substituted aliphatic, optionally substituted heteroaryl, optionally substituted aliphatic, optionally substituted cycloaliphatic or optionally substituted amino; and m is 1 or 2; and each R4x and.R'aX is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4X
and R'ax together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.

[00122] In some other embodiments, R4 is:

Rax~ ,R'ax ~~ ~RSs N
H ~,-Rs9 0 , wherein R58 and R59 are each independently selected from optionally substituted aliphatic, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted (cycloaliphatic)oxy, optionally substituted (heterocycloaliphatic)oxy optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloaliphatic or optionally substituted amino; and each R4x and R'4x is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4x and R'ax together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.
1001231 In several embodiments, R4 is one selected from:
N
O
~N O XN O N~ N N
H H H O H H O \
> > >
O O
O N N N
H H ~ O Li+
`~H O H~N OMe O H O
O
CO2Me +Li-O2C
O H O
O H O AH O H~ NH2 O
`AH O H~N NH2 O HO

O p H
O H 0 `-H H~N NH2 On /
O H p `AH
O p H~N OH
O
H O
4- H H~N NH2 N
O p O / I O

p O O 0 H
N
N
H p H O~ NH2 H p H p OH p sH N
H
> > >
p H O
O p 0 N
N H HO Na+
%)~

p H 0 COZ Na+
> > >
0 O H p `~N N N OMe AH 0 H~N :H2 H H I p l O

H
N
H p H~ NH2 0 0 O N

O N p N O O
N NH H
H O H O 2H H~N NH2 O O
O
O O
H

N N O
H OH H

H O H~ OH
O
N
H
O H O

H HN OBn O O
O ~N CN~ N N
H F2 H H p H
0 p H
p -,~N N
H H OMe H O
H p H N CONH2 O
O EtO2C

O H
0 H O ~N N N
~H H~N p L'+ H O H~ NH2 O p , O O

O O H O H~N

H H'~ NH2 O p O H p H p H~N NHZ "~A 0 N p C02-t-Bu O H 0 ~4H H-'~- N N~ O
H p H NH2 60t-Bu , , O H p H 0 H~N NH p p O ( ~4N C1(N ~,s `'~H NH2 ~ H F2 H `O~ O

~H NHZ H NH2 /H H--~ H NH2 0 f 0 H H ~'H H~ ~H `N
~
O O O H s N ~NH
`HN, " `HN,,, H p H\, ~ H\, ~ v ``'~ N N~
H p O

NN ~ p H O H ~\ OMe / H N-O" ~ ~ O

O
O /

llz~
N OH O H H O H N
N_ N
H O H OMe 0 0 OMe 2rll - ONH
2 H 0 H ~H O H \/ ss``N ZMeO H 0 MeO

~H 0 H O~H N O H \/ /H O H~

O Me0 0 H 2y~,N H N 0 /\ OMe H N O / \ H N
O O H
O NH

H N \ ~,s N N
0 ~O O H O H
_ 0 O \ / N OH
O O
~N N O ~N N/4 H% -H O H H O H

O _ O \ N H O H \/ \
H H H H HN
O O O

_ 4,k p p OMe ~H
H \/
N N~ H N H H 0 p 1 O ~/ p O
O p O T O

H p H H p H ~H p HCF3 H p H~
> > > >
O

O O N 2y~- N
N N p /N N-i ,O H O H
H p H H p H\~~
> > >
0 p / NH
~
H N N p H H
0 H ~\ NJ /N N 0 /
H O H-C MeO
O

O O O O-P
`~ N N `A N N A N N -<---/ H p H p H H p H
> > >
O
J-N O
N
H
H 0 p~xH ~xH OMe ~ O O

, 0 / p ~ ~N 0 ~N N~-O ~N N `H Oo H p H H p H 0 > > >

O
O H p H _ x N p H' pl N ~
H
OH CI
> > >
O
,3s'N 21?- N O N O
H p H ~gH H ~H H~
O O
> >

p ~N_p `AN N ~ ~
0 p H H~ H O H \ I H H
> > >
p O OMe p ~H N~F AH p H-~O~ \ ~H N
2YA, k p H ~ o > > >
~ \
/
O

=
~N 2y-ul lrL, .
H 0 H ~~`~ H N~- ~ N N, /
v > > >
O p OS p H
H o ~ H~~ ~ O O N ~
~ I ~
~ \
N ~ N
N N N
~
H H H H
MeO 0 0 > > >
p 0 F O
-N ~ ~
H N
O H H ~~ F H p H
> > >

O CN p O 2r--' 0 H O H ~/ H NH2 H H
O
II F
F
p 0 N N N
N ~N H p H H p H H p OH

O O O
H N/\ H H Nq O
H O H O
, , [00124] In some specific embodiments, R4 is O NH N p NH2 0 `~ N
--N H

O A o NH NH N H--NH N H
O ~ O 0 , , , F2HC )0'2 F2HC )a2 O '.Z~ ~
NH NH -~-NH NH ~ ~ C(O)-XZOo --or , where X200 is -OX202 or -X202, and X202 is aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl.

O ~
--[00125] In other embodiments, R4 is 0 , H 0 , or [00126] Additional examples of R4 are illustrated in PCT publications WO
2004/103996 Al, WO 2004/72243 A2, WO 03/064456 Al, WO 03/64455 A2, WO 03/064416 Al, and U.S.
Patent Publication US 2005/0090450, as well as those other publications referenced herein, each of which is incorporated in its entirety by reference.
5. Substituent R5i [00127] R5 is -ZER12, wherein each ZE is independently a bond or an optionally substituted branched or straight C1_12 aliphatic chain wherein up to three carbon units of ZD are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NRE-, -C(O)NRENRE-, -C(O)O-, -NREC(O)O-, -0-, -NREC(O)NRE-, -NRENRE-, -S-, -SO-, -SO2-, -NRE-, -S02NRE-, or -NRES02NRE-. Each R12 is independently RE, halo, -OH, -CN, -NO2, -NH2, or -OCF3. Each RE is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[00128] In several embodiments R5 is optionally substituted with 1 to 4 substituents.
[00129] In certain embodiments, R5 1S -Q4-W4-Q3-W3-Q2-W2-Q1; wherein each of W2, W3, and W4 is independently a bond, -C(O)-, -C(S)-, -C(O)N(Q5)-, -C(O)O-, -0-, -N(Qs)C(O)N(Q5)-, -SO2-, -N(Q5)SO2-, -S-, -N(Q5)-, -SO-, -OC(O)-, -N(Q5)C(O)O-, or -SO2N(Q5)-; each of Q1, Q2, Q3 and Q4 is independently a bond, an optionally substituted C14 aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Q1, Q2, Q3, or Q4 is the terminal group of R5; and each Q5 is independently hydrogen or an optionally substituted aliphatic. In some specific embodiments, Q4 is a bond.
[00130] In several embodiments, R5 is an optionally substituted acyl group. In several examples, R5 is an optionally substituted alkylcarbonyl. Additional examples of R5 include (amino)alkylcarbonyl, (halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl, or (heterocycloaliphatic)alkylcarbonyl.
Included in these examples are embodiments where R5 is (heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl, (bicycloaryl(sulfonyl(amino)))alkylcarbonyl, (aryl(alkoxy(carbonyl(amino))))alkylcarbonyl, (alkyl(carbonyl(amino)))alkylcarbonyl, (alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl, (cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl, (alkyl(amino(carbonyl(amino))))alkylcarbonyl, or (bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which is optionally substituted with 1-3 substituents.
[00131] In several embodiments, R5 is an optionally substituted carboxy group.
In one example, R5 is optionally substituted alkoxycarbonyl. Another example of R5 includes C1-4 alkoxycarbonyl, or (tricyclic aryl)alkoxycarbonyl, each of which is optionally substituted with 1-3 substituents. Other carboxy groups include (aliphatic(oxy))carbonyl, a (heteroaralkyl(oxy))carbonyl, (heterocycloaliphatic(oxy)carbonyl, (aralkyl(oxy))carbonyl, each of which is optionally substituted with 1-3 of halo, alkoxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, or combinations thereof.
[00132] In several embodiments, R5 is optionally substituted aminocarbonyl.
Examples of R5 include (alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or (aryl(alkoxy(ca rbonyl(alkyl(amino(carb(5nyl(alkyl)))))))aminocarb6nyl, each of which is optionally substituted with 1-3 substituents.
1001331 In several embodiments, R5 is optionally substituted heteroaryl. In one example, R5 is an optionally substituted oxazolyl, pyrrolyl, furyl, thiophenyl, triazinyl, pyridinyl, pyrazinyl, pyrimidinyl, or pyridazinyl.
[00134] In several embodiments, R5 is an alkylsulfonyl, aminosulfonyl, arylsulfonyl, heteroarylsulfonyl, cycloaliphaticsulfonyl, or heterocycloaliphaticsulfonyl, each of which is optionally substituted with 1-4 substituents.
[00135] In several embodiments, R5 is an optionally substituted alkylsulfonyl.
Examples of R5 include (aryl)alkylsulfonyl, or (alkyl(amino))alkylsulfonyl, each of which is optionally substituted with 1-3 substituents. alkylsulfonyl, aminosulfonyl, arylsulfonyl, -heteroarylsulfonyl, cycloaliphaticsulfonyl, or heterocycloaliphaticsulfonyl, each of which is optionally substituted. In certain embodiments, R5 is an optionally substituted alkylsulfonyl.
[00136] In other certain embodiments, R5 is (aryl)alkylsulfonyl, or (alkyl(amino))alkylsulfonyl, each of which is optionally substituted.

[00137] In some specific embodiments, R5 is (amino)alkylcarbonyl, (halo)alkylcarbonyl, (aryl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl, or (heterocycloaliphatic)alkylcarbonyl, (heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl, (bicycloaryl(sulfonyl(amino)))alkylcarbonyl, (aryl(alkoxy(carbonyl(amino))))alkylcarbonyl, (alkyl(carbonyl(amino)))alkylcarbonyl, (alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl, (cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl, (alkyl(amino(carbonyl(amino))))alkylcarbonyl, or (bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which is optionally substituted.
[00138] In other specific embodiments, R5 is a heteroarylcarbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic(oxy(amido(alkyl))))carbonyl, an (aryl(sulfonyl(amino(alkyl))))carbonyl, an (aralkyl(oxy(amido(alkyl))))carbonyl, an (aliphatic(oxy(amido(alkyl))))carbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic)carbonyl, or a (heteroaryl(amido(alkyl(amido(alkyl))))carbonyl, each of which is optionally substituted with 1-4 of halo, aliphatic, cycloaliphatic, acyl, alkoxy, or combinations thereof.
[00139] In still other embodiments, R5 is amido. For example, R5 is (alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or (aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl, each of which is optionally substituted.
[00140] In several embodiments, R5 is R13 R'13 R13 R 13 R1 3R'1 RT~ O RT~NX /O O O RT
NI S N
RT~ _ R R140 // p~srr~ RT~NRT or O
14 H (R14 2 QI QII QIII QIV QV QVI
wherein T is a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)- or -SO2-; each R
is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R13 and R'13 is independently hydro'gen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R14 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R13 and R14, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic; or R13 and R'13, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic. For clarity, when R5 is QVI, each of R13, R'13 and R14 in each subunit can be independently selected as described above.
The set of R13, R'i3 and R14 variables in one subunit need not necessarily be identical to the same set of R13, R'13 and R14 variables in the other subunit.
[00141] In other embodiments, R5 is QI or QII.
[00142] In some embodiments, R in the substituent in QI, QII, QIIl, QIV, QV, or QVI is ; or [00143] In other embodiments, R5 is QVI and R is O
N

\ A ~
I N `
/ ~ or N
[00144] In other embodiments, R in the substituent in QI, QII, QIIl, QIV, QV, or QVI is R15~ N R15-,,, /N ,KYo N

~ I I R 15 ~ 0 R 15R15 O R'1R15 15 15 H N H N /K N
K n NH K- S~ n N~ R15 \~
p / ~ /
R 0 ~K~ R15 0 /%-K\ R, 15 x R15 HN ~
N
KS
K ( n NH R~ h-NH R/ \\ \ -K ~
X/^\ ~
R 15 O /S -K\ R15 0 /P\\ \ R'15 R15 N H N
K-S ~ n NH K-S~~ " NH R15 K H
/ // O
R15 \O S-K R15 O or 0 R15 0 \ R15 wherein each R15 and R'15 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic, or R15 and R'15 together with the atom to which they are both bound form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic; and each K is independently a bond, C1_12 aliphatic, -0-, -S-, -S(O)2-, -NR16-, -C(O)-, or -C(O)NR16-, wherein R16 is hydrogen or an optionally substituted C1_12 aliphatic; and n is 1-3. For clarity, when more than one R15 is present in QI, QII, QIII, QIV, QV, or QVI, each R15 can be the same or different. In several embodiments, R15 or R'15 is [C3_10 cycloalkyl or C3_10 cycloalkenyl]-C1_12 aliphatic, (3 to 10 membered)-heterocycloaliphatic, (3 to 10 membered)-heterocycloaliphatic-C1_12 aliphatic-, (5 to 10 membered)-heteroaryl, or (5 to 10 membered)-heteroaryl-Ci_1Z aliphatic-.
[00145] In still other embodiments, R in the substituent in QI, QII, QIII, QIV, QV, or QVI is H HN
,N i /O N O N NH ~
~ y 0 _O
\\
OyO O or 0 [00146] In further embodiments, R in the substituent in QI, QII, QIII, QIV, QV, or QVI is - - O~- - S~- - HN~- H - -S~ ~
N N ~ N N O~ N
~/ , O O O O/

N H H H H H I \-- 1)-F I
~ N N > ~
-__ ~ N
N \ N N \N \ ~
\
H ' H H H O H

O

CI DC>-~- N:~ - - \-~-N\ SS ~ y CI \ N N N N H H ' H ' H

\H

Z )n Z p Z )n ( ) Z ( )n Z
\
N~\ S\N \ .
O O N~~
N H H H H H

0 0 p~ /p 0 ~ SO
Z Z~S \
Z )n n SZ Z
n ;I
O~S\ n J~ / ~ O ( ) N~~ O( )NJ\~~
N O N O N H
H H H H

Z n )n Z )n -~ ~ -~ 0'-"~ 1 + Z - -N \ S 0 'N \ N ~
O p H \ H O/ \
H H
0 ~~ \ 0 O
~ ~/'P
Z Z--S Z--S Z-~
)~ N> /N> or \ p S\N
+

H H H \ H

wherein each Z is independently -0-, -S-, -NR50-, or -C(R50)2-, ------ is independently a single bond or a double bond, and each R50 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; and n is 1 or 2.
[00147] In several embodiments, R5 is R'1s R1s N1a 0 R1s R1s R14 0 R14 O
RT-N \
O
RT~ R'13 R13 RT~N R'1'3 R1~ , or R,/13R 13 wherein wherein T is a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)-or -SO2-; each R is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R13 and R'13 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R13 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R13 and R14, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic, in which each heterocycloaliphatic ring; or R13 and R'13, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic; each R15 and R'15 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R15 and R'15 together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring; and each R16 is independently hydrogen or a protecting group.
[00148] In some embodiments, R15 and R'15 together with the atom to which they are attached form a"3 to 7 membered ring. Non-limiting examples include:

cs or [00149] Non-limiting examples of Ri I and R13 include:

i I I ~
~,,,,,,, Jl N
I \ / I \ I \
~ 1-\

O O/Y\
or Alternatively, R13 and R15 together with the atoms to which they are attached may form an optionally substituted 5 to 7 membered monocyclic heterocycloaliphatic or an optionally substituted 6 to 12 membered bicyclic heterocycloaliphatic, in which each heterocycloaliphatic or aryl ring optionally contains an additional heteroatom selected from O,SandN.
[00150] Also, R15 and R16 together to with the atoms to which they are attached can form a ring.

NH
[00151] In several embodiments, R5 is: RT~ , wherein T is -C(O)-, and R is SS`

ell~ OO
o oD
HO O ~.O -S
> > > >

/ ~.

O
or [00152] In several embodiments, R5 is a group selected from:
O
0 I j N 0 H `~.

O O
> > >

O
N (17r OH O ~

H
H (CH3)3C-0~_ , > > >
CN N O
/
O
O N O
H O ~ II O N\ I N 4 HJ~H s^;"' H o > > >

` /

p p O
a ON XN p \/ O p NXN
N H ~N H H
H CI O H
> >
N H O
N N
O O N

O--N O O p H p CH3 > > > >
H O
02S~ N
O O O / O O
~., c, O-~~ O~O~ H N \ p~ H X

O

~ N~~' N
Q-N N H
H H O H H ^;^^' O O O Oq_N O O
N~N O~O~N O N~H X N
H H H H H H
> > > >
p O i 0 NH ~ ~
pN ' ~0 I ~ H p > > > >

H3CN ~2 N N O
H
O CI
> > > >
O O
MeO
I N N~N p O
MeO H H~ '--ON O
OMe H
> > >

O p p O
H N
H H H H p p'J~N 0'11~N I F ~ p~N I
H p I/ H I/ H p \\ ~ ~

rN O O pZN p N -O H
0 j::::N 0 pN 4 p4 NH p 0 H p O
F

\ I 0 0 pc 0 O
N
N H 4 0~H ~`' NH
0 ~ H
O p O
HO'r'-"kH \ ^ ~ ~ p p~H ~
O O TO H
( " O O

N
N x F O H
H

I O FF
p N
H p N

p 0 HN
O N O H
H

, , , 0 ~_\ O
H O

0 \ H 0 p p - O
H H O p NH N
O p H 0 F p 0 N
NJ
p O H p co N H N H 0 _.H O 0 ~

p ~ 0 N
N ~+ N ~+
p I~ p H 0 H 0 ~ H 0 0 ~' 0 HN N
H O ~-NH H H

N H
N

F

WO 2008/106058 PCT/US2008/002395 1-1 ll.~

0 O~H ~ H ~
~N 0 p~z,~ O 0 ~, O

(?"'~ H 0 O
0 NN 0 N'N H O
O, N H 0 H

N
i H 0 0 N
N 0"'~~
H
H p N 0 , , , HO O "O
O 0 ~N
N H N H
N - 4j: __F

/p~ I O Br \ / NH 0~ I 0 N O N

O
H HZNN~ H N ~ H

C_N/ 0 H 0 N N N

N H H O
0 ~
~
p p H N

, , O .p N
N
H
O O
N N Y\
H

N

O
p p O H
O~ 0 X H '~ - 0 p CI ~
~ O 0 N

H O~pXH , "` H 0 0 ~
ANH

H N
p 71', -0 H <2 S02 0 )FS 0 ~ 0 N H

0 H ~' 0 0 ,~,O
O
HN
X HN
0 p O
O
N O

H ~ 0 p O 6\--p`~^ 0X H ~N

\ O\S N O

H N N
I / O H H

0 p N~ ' 011 O
O~N 0 H I N- N
H ~ ~ >-H H

O

p N--~,N
O OXN, N H H

~ H H ~ O

N

H H
fo O
H H

O
H

p HO
O
~Q~ '- --O
e N
H ~,and O
[00153] In some embodiments, R5 is Xi oo Qr0?
H O

02 Xloo = NH, CH2 X99 = OR, OC(NH)R, or R-S, N=~
where R is defined above.

[00154] Additional examples of R5 are illustrated in PCT publications WO
2004/103996 Al, WO 2004/72243 A2, WO 03/064456 Al, WO 03/64455 A2, WO 03/064416 A1, and U.S.
Patent Publication US 2005/0090450, as well as those other publications referenced herein, each of which is incorporated in its entirety by reference.
C. Sub-generic Compounds:
[00155] Another aspect of the present invention provides compounds of formula la useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula la include:

~~
.~iRs N\
A
N
R5 a R4a O
Ia or a pharmaceutically acceptable salt thereof wherein RI, R2, R3, and A are defined above in formula I.
[00156] Each R4a is -Z1-V1-Z2-V2-Z3-V3 each of Vi, V2, and V3 is independently a bond, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Vl, V2, V3 is the terminal group of R2; each of Zl, Z2, and Z3 is independently a bond, -C(O)-, -C(O)C(O)-, -C(S)-, -C(O)N(Q5)-, -N(Q5)C(O)-, -C(O)C(O)N(QS)-, -0-, , SO-, -SO2-, -N(Q5)SO2-, -N(Q5)C(O)N(Q5)-, -N(Q5)C(S)N(Q5)-, -N(Q5)-, -N(Q5)S02-, -SO2N(Q5)-, -C(O)N(Q5)S02-, -SO2N(Q5)C(O)-, or hydrogen when Zl, Z2, or Z3 is the terminal group of R2; and each Q5 is independently hydrogen, or an optionally substituted aliphatic.

[00157] Each R5a 1S -Q4-W4-Q3-W3-Q2-W2-Ql; wherein each of W2, W3, and W4 is independently a bond, -C(O)-, -C(S)-, -C(O)N(Q5)-, -C(O)O-, -0-, -N(Q5)C(O)N(Q5)-, -SO2-, -N(Q5)S02-, -S-, -N(Q5)-, -SO-, -N(Q5)C(O)-, -OC(O)-, -N(Q5)C(O)O-, or -SO2N(Q5)-; each of Ql, Q2, Q3 and Q4 is independently a bond, an optionally substituted C14 aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Ql, Q2, Q3, or Q4 is the terminal group of R5a; and each Q5 is independently hydrogen or an optionally substituted aliphatic.
[00158] In several examples, R4a is an optionally substituted (aliphatic)amino, an optionally substituted alkoxy, or hydroxy.
1001591 In several examples, R4a is an (aliphatic)amino wherein the nitrogen atom is optionally substituted with -Z2-V2-Z3-V3 or =Z3-V3 wherein each of Z2 and Z3 is independently a bond, -C(O)-, -N(Q5)-, or -C(O)C(O)N(Q5)-; and each of V2 and V3 is independently a bond, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic.
[00160] Another aspect of the present invention provides compounds of formula Ib useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula Ib include:

R Rz ~N R3 A

O N O
R-T-N
I

ro or a pharmaceutically acceptable salt thereof, wherein Rl, R2, R3, R14, R, T, and A are defined above in formula I.
[00161] Each G is a 2 to 15 atom optionally substituted aliphatic chain optionally containing 1 to 3 heteroatoms selected from 0, S and N.
[00162] Examples of compounds of formula Ib include:

R'\N-KA R14 H O OH
H O OOSiO R

~ N H \T~ O
R-T~N\ O
O O
or wherein Ri, R2, R3, R8, R, T, and A are defined above in formula I.
[00163] Still other examples of formula Ib are R, N R2 R, ~R3 N R2 A
O N
RT,,, N::r NH RT,,,, N
~
O O N O N H

NH O H
O N
~
R4w O R4w or R~

Rs A
O N
RT~
NH
O O

O .\
N--R4w wherein Ri, R2, R3, and A are defined in formula I
,.~CH3, 'Z~CFi3 , above, and each R4W is independently or hydrogen; each T is independently a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)-, or -SO2-; each R is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R14 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl.
[00164] Further specific examples of compounds of formula lb are CI I? CI
O
~N N--r O

N-X NH
H O O
0 H 0 NH ~
N-Q O
p H or ~ H~
[00165] Other examples of compounds of formula Ib include:
CI
O ~ N bN)"O
CI H O H O
N N N OH `_O~N tN N OH
O JI O
O O O O
> >
CI

N )(0 H O
H 0 0, t~~
~O~N N O N H.SO N N O N H.SO

O ~
~ N
I O CI ~)N O
/ N H O
ON N O OH C N N N H-So O O O
~ ~ o O
or [00166] Another aspect of the present invention provides compounds of formula II useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula II include:

Ria\ O
N-~
A

O N O
N\ O R4X R4z R5b R74 R 4x N\

II
or a pharmaceutically acceptable salt thereof, wherein R4x, R'4x, R4z, R4w, and A are defined above.
[00167] Each Ria is an optionally substituted aryl or an optionally substituted heteroaryl.
[00168] Each R4Y is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.
[00169] Each R14 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic.
[00170] Each R4x and R'4x is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R4x and R'4x together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring, or R4x and R4Y together with the atoms to which they are attached form an optionally substituted 5 to 7 membered heterocycloaliphatic ring.
[00171] Each R5b is -ZFR21, wherein ZF is -CH2-, -NH-, -CH(R22)-, or -0-, and R21 is optionally substituted 6-7 membered cycloaliphatic or optionally substituted tert-butyl; and R22 is optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, optionally substituted aryl , or optionally substituted heteroaryl.
[00172] Each R4z is independently hydrogen, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, or optionally substituted aliphatic.

[00173] Additionally, each R4w is independently hydrogen, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, or optionally substituted aliphatic, or R4z and R4w, together with the nitrogen atom to which they are attached form an optionally substituted heterocycloaliphatic.
[00174] Another aspect of the present invention provides compounds of formula III useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula III include:

R1e O
O

A

O
N R4e R H N~R'4e Se N
O H O H

III
or a pharmaceutically acceptable salt thereof, wherein A is defined above in formula I and . o O
R5e lS > > > > > >
SS` O
o HO
O > > > or [00175] R4e is:

, F
~~
or [00176] R'4e is:

, , CH3 , %~'.'CH3 ~
or hydrogen; and Rie is optionally substituted aryl or optionally substituted heteroaryl.
[00177] Another aspect of the present invention provides compounds of formula IV useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula IV include:

Rit R'it 0 A

Rae N ,R'ae R$e H 0 N H

IV
or a pharmaceutically acceptable salt thereof, wherein R5e is:

o cr O
0 , HO
or F
i'~/CH3 F
~
or [00178] R4e 1S

~CH3 ~ 'CH3 [00179] R'4e is or hydrogen.
[001801 Additionally, each of Rif and R'i f is independently hydrogen, sulfonamide, sulfonyl, sulfinyl, optionally substituted acyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, optionally substituted aryl, or optionally substituted heteroaryl, or Rlf and R'if together with the nitrogen atom to which they are attached form an optionally substituted, saturated, partially unsaturated, or full unsaturated, 5-8 membered heterocycloaliphatic or heteroaryl.
[00181] Another aspect of the present invention provides compounds of formula V useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula V include:

D/ D\D
DzzzzD
\ N

A
O O
N R4e ~-N R'4e R5e H N H
O H O
V
or a pharmaceutically acceptable salt thereof, wherein Rsej R4e, and R'4e are defined above in formula III.
[00182] Each D is independently -CR14-, N, S, or 0, provided that no more than two D are independently, S, or 0; and R14 is defined above in formula I.
[00183] Another aspect of the present invention provides compounds of formula VI useful for inhibiting serine protease activity and methods inhibiting serine protease activity.
Compounds of formula VI include:

Rlg\ O
N
A
N
O NH O
NH O O R'4e R4e N
R5e H
VI
or a pharmaceutically acceptable salt thereof, wherein Rse, R4e, and R'4e are defined above in formula III.

[00184] Each Rig is a substituted aryl or a substituted heteroaryl. In some embodiments, Rig O~ O 0 CI
CI \ CI N CI N gnN

,nr ,nr ,ni=
is O~ I \ O CI F CI
1 10 t, ,nr I / O~
O/ ~ .nr > > > > >
O--\

O CI O 0 CI ci:10 .1J' .nr nr i ~
, , , or D. Combinations of Embodiments [00185] Other embodiments include any combination of the aforementioned substituents R1, R2, R3, R4, R5, and A.
E. Exemplary Compounds [00186] The invention is intended to include compounds wherein Ri and R2 contain structural elements of a serine protease inhibitor. Compounds having the structural elements of a serine protease inhibitor equivalent to substituents R, and R2 include, but are not limited to, the compounds of the following publications: WO 97/43310, US 20020016294, WO
01/81325, WO 01/58929, WO 01/32691, WO 02/08198, WO 01/77113, WO 02/08187, WO 02/08256, WO 02/08244, WO 03/006490, WO 01/74768, WO 99/50230, WO 98/17679, WO 02/48157, WO 02/08251, WO 02/07761, WO 02/48172, WO 02/08256, US
20020177725, WO 02/060926, US 20030008828, WO 02/48116, WO 01/64678, WO
01/07407, WO 98/46630, WO 00/59929, WO 99/07733, WO 00/09588, US 20020016442, WO 00/09543, WO 99/07734, US 6,018,020, US 6,265,380, US 6,608,027, US
20020032175, US 20050080017, WO 98/22496, WO 05/028502, US 5,866,684, WO 02/079234, WO 00/31129, WO 99/38888, WO 99/64442, WO 2004072243, WO
02/18369, US2006046956, US2005197301, W02005058821, W02005051980, WO2005030796, WO2005021584, W02005113581, W02005087731, W02005087725, W02005087721, W02005085275, W02005085242, US2003216325, W02003062265, W02003062228, W02002008256, WO 2002008198, W02002008187, WO 2002048172, WO 2001081325, WO 2001077113, US 6251583, US 5990276, US20040224900, US20040229818, W02004037855, W02004039833, W0200489974, W02004103996, W02004030670, W02005028501, W02006007700, W02005070955, W02006007708, W02006000085, W02005073195, W02005073216, W02004026896, W02004072243, W02004113365, W02005010029, US20050153877, W02004093798, W02004094452, W02005046712, W02005051410, W02005054430, W02004032827, W02005095403, W02005077969, W02005037860, W02004092161, W02005028502, W02003087092, and W02005037214, each of which is incorporated herein by reference.
[001871 Specific exemplary compounds of the invention are shown below in Table 1.

Table 1: Exemplary compounds of the present invention.

~ CI
o N~ O
N
`

0 H 0 O j~ ~ NH 0-11H O 0 ~ NH
b N o N O
O

a-~H O O 0 NH ~'O H O O ji NH
~ b b ci N-~ O
,10 N
~~.

Q~H O 0 ~ NH O-~H 0 0 ~ NH
b b /

CI C) .O o N ~

O

a0 H C C ~ NH ON N O
H 0 ~N H
~ 0-~-~ b CI
\ ~ O
N-~
,,\0 O t.N N

III. SYNTHETIC SCHEMES
[00188] Compounds of Formula I may be readily synthesized from commercially available starting materials using the exemplary synthetic routes provided below.
Exemplary synthetic routes to produce compounds of Formula I are provided below in the Preparations, Methods, Examples, and Schemes.
[00189] A method for preparing compounds of formula I, wherein A is 0, is illustrated in Scheme 1.

Scheme 1:

0 H OEt HN 0 R, . ~0 i8 O Ib ic p d N p N
.
Boc 0 Boc 0r Boc 0 0r BocN O
1a 1b 1c 1d R, \ N --~ 0 R, \N ~O R, \ N--~ O

0 ie 0 if 0 ig HN 0 RT,, N 'j~ ~/N 0 RT-1 NN OH

1e 1f 1g R--f 0 RN--f 0 0 i h O
RT R13 N Rav OH /R4W RT R13 N R4Y 0 ~R4w ,,N N N` ,,N~{ N N`
H O 0 R4z H \\O O Raz 1h I
[00190] The diprotected prolineone 1 a reacts with ethyl bromoacetate in the presence of zinc and trimethylsilyl chloride to produce the ester intermediate lb as a 4:1 diastereomeric mixture. The ester intermediate 1 b reacts with hydrazine to form an intermediate hydrazide (not shown) which in turn is oxidized with, for example, sodium nitrite followed by cyclization to produce the substituted spiropyrrolidine intermediate 1 c. The substituted spiropyrrolidine intermediate 1 c can be converted to the substituted intermediate 1 d by two methods. Reaction of 1 c with an aryl halide such as, for example, a substituted bromobenzene in the presence of a palladium catalyst such as, for example, PdZ(dba)3, Xantphos and potassium carbonate provides intermediates ld wherein R, is aryl.
Alternatively, intermediate 1 c may be alkylated with an appropriate alkyl halide such as, for example, benzyl bromide or ethyl iodide in the presence of a base such as, for example, sodium hydride to provide intermediates ld wherein Ri is an optionally substituted alkyl.
The diastereomers of I d may be separated using chromatography.
[00191] Selective removal of the N protecting group from 1 d to provide 1 e may be accomplished with HCl in ethyl acetate. The intermediates 1 e are further converted to the intermediate lf by reaction of le with R12-C(O)-OH in the presence of a coupling reagent such as, f o r example, EDC, HOBT and DIEA. Removal of the ester protecting group f r o m 1 f using, for example, TFA in methylene chloride, provides the acid intermediate 1 g. Reaction of I g with RI i-N(R )NH in the presence of a coupling reagent such as, for example, EDC, HOBT and DIEA provides compounds of the invention wherein A is O. In certain embodiments, R< < may contain the hydroxy moiety -CH(OH)C(O)-, oxidation of which with, for example, Des-Martin periodinane or sodium hypochlorite in the presence of TEMPO provides compounds of formula I wherein R12 contains the moiety -C(O)C(O)-.
[001921 Scheme 2:

O Et Et N iia iib iic Boc 0 Boc N O ~N
O Boc O
O O
1a 2a 2b H2N OEt H2N OH HN O
O O
iid iie N
Boc N O Boc N O Boc O
O O O
2c 2d 2e [00193] Referring to Scheme 2, the diprotected prolineone 1 a reacts with (carboethoxy-methylene)triphenylphosporane to provide the unsaturated ester 2a. Reaction of 2a with nitro methane in the presence of tetramethylguanidine (TMG) provides the intermediate nitro compound 2b. Catalytic hydrogenation of 2b in the presence of 10% Pd/C
provides the amino compound 2c. Hydrolysis of 2c with lithium hydroxide in water provides the acid 2d which is cyclized in the presence of EDC, HOBT and DIEA to provide the lactam intermediate 2e. Conversion of the intermediate 2e to compounds of formula I
wherein A is CH2 is achieved as previously described under Scheme I for I c.
IV. FORMULATIONS, ADMINISTRATIONS, AND USES
1001941 Another embodiment of this invention provides a pharmaceutical composition comprising a compound of Formula I or pharmaceutically acceptable salts or mixtures of salts thereof. According to another embodiment, the compound of Formula I is present in an amount effective to decrease the viral load in a sample or in a patient, wherein said virus encodes a serine protease necessary for the viral life cycle, and a pharmaceutically acceptable carrier.
[00195] If pharmaceutically acceptable salts of the compounds of this invention are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2 naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 phenyl propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N methyl D glucamine, and salts with amino acids such as arginine, lysine, and so forth.
[00196] Also, the basic nitrogen containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides;
dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil soluble or dispersible products are thereby obtained.
[00197] The compounds utilized in the compositions and methods of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
[00198] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat.
[00199] According to another embodiment, the compositions of this invention are formulated for pharmaceutical administration to a mammal. In one embodiment said mammal is a human being.
[00200] Such pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra articular, intra synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally or intravenously.
[00201] Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
1002021 In one embodiment, dosage levels of between about 0.01 and about 100 mg/kg body weight per day of the protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of antiviral, particularly anti-HCV mediated disease. In another embodiment, dosage levels of between about 0.5 and about 75 mg/kg body weight per day of the protease inhibitor compounds described herein are useful in a monotherapy for the prevention and treatment of antiviral, particularly anti-HCV mediated disease. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95% active compound (w/w). In one embodiment, such preparations contain from about 20% to about 80% active compound.
[00203] When the compositions of this invention comprise a combination of a compound of formula I and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 10 to 100% of the dosage normally administered in a monotherapy regimen. In another embodiment, the additional agent should be present at dosage levels of between about 10 to 80%
of the dosage normally administered in a monotherapy regimen.
[00204] The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
[00205] Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the agent with a suitable non irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
[00206] The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract.
Suitable topical formulations are readily prepared for each of these areas or organs.
[00207] Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.
Topically transdermal patches may also be used.

[00208] For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.
[00209] For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
[00210] The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
[00211] In one embodiment, the pharmaceutical compositions are formulated for oral administration.
[00212] In another embodiment, the compositions of this invention additionally comprise another anti-viral agent, preferably an anti-HCV agent. Such anti-viral agents include, but are not limited to, immunomodulatory agents, such as a, (3-, and y-interferons, pegylated derivatized interferon-a compounds, and thymosin; other anti-viral agents, such as ribavirin, amantadine, and telbivudine; other inhibitors of hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including helicase and polymerase inhibitors; inhibitors of internal ribosome entry;
broad-spectrum viral inhibitors, such as IMPDH inhibitors (e.g., compounds of U.S. Pat. Nos.
5,807,876, 6,498,178, 6,344,465, and 6,054,472, WO 97/40028, WO 98/40381, WO 00/5633 1, and mycophenolic acid and derivatives thereof, and including, but not limited to VX-497, VX-148, and/or VX-944; or combinations of any of the above. See also W. Markland et al., Antimicrobial & Antiviral Chemotherapy, 44, p. 859 (2000) and U.S. Pat. No.
6,541,496.

O N N N O O H

[00213] The following definitions are used herein (with trademarks referring to products available as of this application's filing date).
[00214] "Peg-Intron" means PEG-INTRON , peginteferon alfa-2b, available from Schering Corporation, Kenilworth, NJ.
[00215] "Intron" means INTRON-A , interferon alfa-2b available from Schering Corporation, Kenilworth, NJ.
[00216] "Ribavirin" means ribavirin (1-beta-D-ribofuranosyl-lH-1,2,4-triazole-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, CA;
described in the Merck Index, entry 8365, Twelfth Edition; also available as REBETROL from Schering Corporation, Kenilworth, NJ, or as COPEGASUS from Hoffmann-La Roche, Nutley, NJ.
1002171 "Pagasys" means PEGASYS , peginterferon alfa-2a available Hoffmann-La Roche, Nutley, NJ.
[00218] "Roferon" mean ROFERON , recombinant interferon alfa-2a available from Hoffmann-La Roche, Nutley, NJ.
[00219] "Berefor" means BEREFOR , interferon alfa 2 available from Boehringer Ingelheim.Pharmaceutical, Inc., Ridgefield, CT.
[00220] SUMIFERON , a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan.
[00221] WELLFERON , interferon alpha nl available from Glaxo_Wellcome LTd., Great Britain.
[00222] ALFERON , a mixture of natural alpha interferons made by Interferon Sciences, and available from Purdue Frederick Co., CT.
1002231 The term "interferon" as used herein means a member of a family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation, and modulate immune response, such as interferon alpha, interferon beta, or interferon gamma. The Merck Index, entry 5015, Twelfth Edition.
[00224] According to one embodiment of the present invention, the interferon is a-interferon. According to another embodiment, a therapeutic combination of the present invention utilizes natural alpha interferon 2a. Or, the therapeutic combination of the present invention utilizes natural alpha interferon 2b. In another embodiment, the therapeutic combination of the present invention utilizes recombinant alpha interferon 2a or 2b. In yet another embodiment, the interferon is pegylated alpha interferon 2a or 2b.
Interferons suitable for the present invention include:
(a) INTRON-A (interferon-alpha 2B, Schering Plough), (b) PEG-INTRON , (c) PEGASYS , (d) ROFERON , (e) BEREFOR , (f) SUMIFERON , (g) WELLFERON , (h) consensus alpha interferon available from Amgen, Inc., Newbury Park, CA, (i) ALFERON ;
(j) VIRAFERON ;
(k) INFERGEN ;
(1) ALBUFERONTM
[00225] As is recognized by skilled practitioners, a protease inhibitor would be preferably administered orally. Interferon is not typically administered orally.
Nevertheless, nothing herein limits the methods or combinations of this invention to any specific dosage forms or regime. Thus, each component of a combination according to this invention may be administered separately, together, or in any combination thereof.
[00226] In one embodiment, the protease inhibitor and interferon are administered in separate dosage forms. In one embodiment, any additional agent is administered as part of a single dosage form with the protease inhibitor or as a separate dosage form.
As this invention involves a combination of compounds, the specific amounts of each compound may be dependent on the specific amounts of each other compound in the combination.
As recognized by skilled practitioners, dosages of interferon are typically measured in IU (e.g., about 4 million IU to about 12 million IU).
[00227] Accordingly, agents (whether acting as an immunomodulatory agent or otherwise) that may be used in combination with a compound of this invention include, but are not limited to, AlbuferonTM (albumin-Interferon alpha) available from Human Genome Sciences;
PEG-INTRON (peginterferon alfa-2b, available from Schering Corporation, Kenilworth, NJ); INTRON-A , (interferon alfa-2b available from Schering Corporation, Kenilworth, NJ); ribavirin (1-beta-D-ribofuranosyl-lH-1,2,4-triazole-3-carboxamide, available from ICN
Pharmaceuticals, Inc., Costa Mesa, CA; described in the Merck Index, entry 8365, Twelfth Edition); REBETROL (Schering Corporation, Kenilworth, NJ), COPEGUS (Hoffmann-La Roche, Nutley, NJ); PEGASYS (peginterferon alfa-2a available Hoffinann-La Roche, Nutley, NJ); ROFERON (recombinant interferon alfa-2a available from Hoffmann-La Roche, Nutley, NJ); BEREFOR (interferon alfa 2 available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT); SUMIFERON (a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan); WELLFERON
(interferon alpha nl available from Glaxo Wellcome Ltd., Great Britain); ALFERON (a mixture of natural alpha interferons made by Interferon Sciences, and available from Purdue Frederick Co., CT); a-interferon; natural alpha interferon 2a; natural alpha interferon 2b; pegylated alpha interferon 2a or 2b; consensus alpha interferon (Amgen, Inc., Newbury Park, CA);
VIRAFERON ; INFERGEN ; REBETRON (Schering Plough, Interferon-alpha 2B +
Ribavirin); pegylated interferon alpha (Reddy, K.R. et al. "Efficacy and Safety of Pegylated (40-kd) Interferon alpha-2a Compared with Interferon alpha-2a in Noncirrhotic Patients with Chronic Hepatitis C (Hepatology, 33, pp. 433-438 (2001); consensus interferon (Kao, J.H., et al., "Efficacy of Consensus Interferon in the Treatment of Chronic Hepatitis"
J.
Gastroenterol. Hepatol. 15, pp. 1418-1423 (2000); lymphoblastoid or "natural"
interferon;
interferon tau (Clayette, P. et al., "IFN-tau, A New Interferon Type I with Antiretroviral activity" Pathol. Biol. (Paris) 47, pp. 553-559 (1999); interleukin-2 (Davis, G.L. et al., "Future Options for the Management of Hepatitis C." Seminars in Liver Disease, 19, pp. 103-112 (1999); =Interleukin-6 (Davis et al. "Future Options for the Management of Hepatitis C."
Seminars in Liver Disease, 19, pp. 103-112 (1999); interleukin-12 (Davis, G.L.
et al., "Future Options for the Management of Hepatitis C." Seminars in Liver Disease, 19, pp.

(1999); and compounds that enhance the development of type 1 helper T cell response (Davis et al., "Future Options for the Management of Hepatitis C." Seminars in Liver Disease, 19, pp. 103-112 (1999)). Also included are compounds that stimulate the synthesis of interferon in cells (Tazulakhova, E.B. et al., "Russian Experience in Screening, analysis, and Clinical Application of Novel Interferon Inducers" J. Interferon Cytokine Res., 21 pp.
65-73) including, but are not limited to, double stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M Pharmaceuticals; Sauder, D.N. "Immunomodulatory and Pharmacologic Properties of Imiquimod" J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).
J002281 Compounds that stimulate the synthesis of interferon in cells (Tazulakhova, E.B. et al., "Russian Experience in Screening, analysis, and Clinical Application of Novel Interferon Inducers" J. Interferon Cytokine Res., 21 pp. 65-73) include, but are not limited to, double stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M

Pharmaceuticals; Sauder, D.N. "Immunomodulatory and Pharmacologic Properties of Imiquimod" J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).
[00229] Other non-immunomodulatory or immunomodulatory compounds may be used in combination with a compound of this invention including, but not limited to, those specified in WO 02/18369, which is incorporated herein by reference (see, e.g., page 273, lines 9-22 and page 274, line 4 to page 276, line 11).
[00230] Still other agents include those described in various published U.S.
Patent Applications. These publications provide additional teachings of compounds and methods that could be used in combination with N-(3-(3-(3-methoxy-4-(oxazol-5-yl)phenyl)ureido)benzyl)tetrahydrofuran-3-carboxamide (VX950) in the methods of this invention, particularly for the treatment of hepatitis. It is contemplated that any such methods and compositions may be used in combination with the methods and compositions of the present invention. For brevity, the disclosure the disclosures from those publications is referred to be reference to the publication number but it should be noted that the disclosure of the compounds in particular is specifically incorporated herein by reference.
Exemplary such publications include U.S. Patent Publication No. 20040058982; U.S. Patent Publication No.
20050192212; U.S. Patent Publication No. 20050080005; U.S. Patent Publication No.
20050062522; U.S. Patent Publication No. 20050020503; U.S. Patent Publication No.
20040229818; U.S. Patent Publication No. 20040229817; U.S. Patent Publication No.
20040224900; U.S. Patent Publication No. 20040186125; U.S. Patent Publication No.
20040171626; U.S. Patent Publication No. 20040 1 1 0747; U.S. Patent Publication No.
20040072788; U.S. Patent Publication No. 20040067901; U.S. Patent Publication No.
20030191067; U.S. Patent Publication No. 20030187018; U.S. Patent Publication No.
20030186895; U.S. Patent Publication No. 20030181363; U.S. Patent Publication No.
20020147160; U.S. Patent Publication No. 20040082574; U.S. Patent Publication No.
20050192212; U.S. Patent Publication No. 20050187192; U.S. Patent Publication No.
20050187165; U.S. Patent Publication No. 20050049220; and U.S. Patent Publication No.
US2005/0222236.
[00231] This invention may also involve administering a cytochrome P450 monooxygenase inhibitor. CYP inhibitors may be useful in increasing liver concentrations and/or increasing blood levels of compounds that are inhibited by CYP.
[00232] If an embodiment of this invention involves a CYP inhibitor, any CYP
inhibitor that improves the pharmacokinetics of the relevant NS3/4A protease may be used in a method of this invention. These CYP inhibitors include, but are not limited to, ritonavir (WO

94/14436), ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole, cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin,VX-944, and VX-497. Preferred CYP inhibitors include ritonavir, ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, and clomethiazole. For preferred dosage forms of ritonavir, see U.S. Pat. No. 6,037, 157, and the documents cited therein: U.S. Pat. No. 5,484,801, U.S. Application Serial No. 08/402,690, WO
95/07696 and WO 95/09614.
[00233] Methods for measuring the ability of a compound to inhibit cytochrome monooxygenase activity are known. See, e.g., U.S. Pat. No. 6,037,157, and Yun, et al. Drug Metabolism & Disposition, vol. 21, pp. 403-407 (1993).
1002341 Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary.
Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease.
Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
[00235] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredients will also depend upon the particular described compound and the presence or absence and the nature of the additional anti-viral agent in the composition.
[00236] According to another embodiment, the invention provides a method for treating a patient infected with a virus characterized by a virally encoded serine protease that is necessary for the life cycle of the virus by administering to said patient a pharmaceutically acceptable composition of this invention. In one embodiment, the methods of this invention are used to treat a patient suffering from a HCV infection. Such treatment may completely eradicate the viral infection or reduce the severity thereof. In another embodiment, the patient is a human being.
1002371 In an alternate embodiment, the methods of this invention additionally comprise the step of administering to said patient an anti-viral agent preferably an anti-HCV agent. Such anti-viral agents include, but are not limited to, immunomodulatory agents, such as a-, (3-, and y-interferons, pegylated derivatized interferon-a compounds, and thymosin;
other anti-viral agents, such as ribavirin, amantadine, and telbivudine; other inhibitors of hepatitis C
proteases (NS2-NS3 inhibitors and NS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle, including but not limited to helicase and polymerase inhibitors; inhibitors of internal ribosome entry; broad-spectrum viral inhibitors, such as IMPDH
inhibitors (e.g., VX-497 and other IMPDH inhibitors disclosed in U.S. Pat. Nos. 5,807,876 and 6,498,178, mycophenolic acid and derivatives thereof); inhibitors of cytochrome P-450, such as ritonavir, or combinations of any of the above.
1002381 Such additional agent may be administered to said patient as part of a single dosage form comprising both a compound of this invention and an additional anti-viral agent.
Alternatively the additional agent may be administered separately from the compound of this invention, as part of a multiple dosage form, wherein said additional agent is administered prior to, together with or following a composition comprising a compound of this invention.
[00239] Pharmaceutical compositions may also be prescribed to the patient in "patient packs"
containing the whole course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patients supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in traditional prescriptions.
The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.
[00240] It will be understood that the administration of the combination of the invention by means of a single patient pack, or patient packs of each formulation, containing within a package insert instructing the patient to the correct use of the invention is a desirable additional feature of this invention.
[00241] According to a further aspect of the invention is a pack comprising at least one compound of formula I (in dosages according to this invention) and an information insert containing directions on the use of the combination of the invention. Any composition, dosage form, therapeutic regimen or other embodiment of this invention may be presented in a pharmaceutical pack. In an alternative embodiment of this invention, the pharmaceutical pack-further comprises one or more of additional agent as described herein.
The additional agent or agents may be provided in the same pack or in separate packs.
[00242] Another aspect of this involves a packaged kit for a patient to use in the treatment of HCV infection or in the prevention of HCV infection (or for use in another method of this invention), comprising: a single or a plurality of pharmaceutical formulation of each pharmaceutical component; a container housing the pharmaceutical formulation(s) during storage and prior to administration; and instructions for carrying out drug administration in a manner effective to treat or prevent HCV infection.
[00243] Accordingly, this invention provides kits for the simultaneous or sequential administration of a dose of at least one compound of formula I (and optionally an additional agent). Typically, such a kit will comprise, e.g. a composition of each compound and optional additional agent(s) in a pharmaceutically acceptable carrier (and in one or in a plurality of pharmaceutical formulations) and written instructions for the simultaneous or sequential administration.
[00244] In another embodiment, a packaged kit is provided that contains one or more dosage forms for self administration; a container means, preferably sealed, for housing the dosage forms during storage and prior to use; and instructions for a patient to carry out drug administration. The instructions will typically be written instructions on a package insert, a label, and/or on other components of the kit, and the dosage form or forms are as described herein. Each dosage form may be individually housed, as in a sheet of a metal foil-plastic laminate with each dosage form isolated from the others in individual cells or bubbles, or the dosage forms may be housed in a single container, as in a plastic bottle. The present kits will also typically include means for packaging the individual kit components, i.e., the dosage forms, the container means, and the written instructions for use. Such packaging means may take the form of a cardboard or paper box, a plastic or foil pouch, etc.
[00245] A kit according to this invention could embody any aspect of this invention such as any composition, dosage form, therapeutic regimen, or pharmaceutical pack. The packs and kits according to this invention optionally comprise a plurality of compositions or dosage forms. Accordingly, included within this invention would be packs and kits containing one composition or more than one composition.
[00246] In yet another embodiment the present invention provides a method of pre-treating a biological substance intended for administration to a patient comprising the step of contacting said biological substance with a pharmaceutically acceptable composition comprising a compound of this invention. Such biological substances include, but are not limited to, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like;
organs such as kidney, liver, heart, lung, etc; sperm and ova; bone marrow and components thereof, and other fluids to be infused into a patient such as saline, dextrose, etc.
[00247] According to another embodiment the invention provides methods of treating materials that may potentially come into contact with a virus characterized by a virally encoded serine protease necessary for its life cycle. This method comprises the step of contacting said material with a compound according to the invention. Such materials include, but are not limited to, surgical instruments and garments (e.g. clothes, gloves, aprons, gowns, masks, eyeglasses, footwear, etc.); laboratory instruments and garments (e.g.
clothes, gloves, aprons, gowns, masks, eyeglasses, footwear, etc.); blood collection apparatuses and materials;
and invasive devices, such as, for example, shunts and stents.
'[00248] In another embodiment, the compounds of this invention may be used as laboratory tools to aid in the isolation of a virally encoded serine protease. This method comprises the steps of providing a compound of this invention attached to a solid support;
contacting said solid support with a sample containing a viral serine protease under conditions that cause said protease to bind to said solid support; and eluting said serine protease from said solid support.
In one embodiment, the viral serine protease isolated by this method is HCV

protease.
1002491 All references cited within this document are incorporated herein by reference.
V. PREPARATIONS AND EXAMPLES
1002501 In order that the invention described herein may be more fully understood, the following preparations and examples are provided. It should be understood that these methods and examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
A. Preparation of Intermediates for Compounds of Formula I
[00251] Set forth below are various methods for preparing intermediates that can be used to synthesize the compound of Formula I.
[00252] Set forth below are various methods for preparing intermediates that can be used to synthesize the compound of Formula I.

HN O
Cbz HO OH
Preparation of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid.
[00253] A solution of the cyanohydrin prepared according to methods described in WO
04/113294 (1 g, 3.65 mmol) in conc. HCI (12 mL) was heated to reflux for 18 hrs. The reaction was concentrated in vacuo to afford the desired amino acid as an HC1 salt (1.7 g) which was used in the next step without further purification. A solution of the above HC1 salt in THF was treated with DIPEA (2.68 g) and Z-OSu (5.16 g). The reaction mixture was stirred at room temperature for 8 hrs. The reaction mixture was diluted with toluene and HC1 (12 N, until pH = 1). After separation, the organic layer was extracted with sat. NaHCO3 (50 mL, twice). The aqueous layer was made acidic with HC1(6 N) until pH = 1 and extracted with EtOAc (200 mL). The combined organic layer was dried and concentrated in vacuo to afford the title compound (0.6 g). (M+1) 308.

HN O
Cbz HO HN-Preparation of benzyl 1-cyclobutyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate.
[00254] To a solution of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (250 mg, 0.81 mmol) in DCM (20 mL) was added HOSu (140 mg, 1.22 mmol), EDC
(234 mg, 1.22 mmol). After stirring for 1 hr, methylamine in THF (2 N, 0.81 mL) was added to the above mixture. The reaction mixture was stirred for 18 hrs and then concentrated in vacuo. The residue was purified by Gilson Prep to afford the title compound (135 mg). 1H-NMR (CDC13): S 7.54-7.28 (m, 5H), 6.67 (NH, 1 H), 5.03 (dd, 2H), 3.68 (m, 1 H), 2.73 (m, 3H), 2.26 (m, IH), 1.97-1.31 (m, 9H). (M+1) 321.

Cbz HO HN--a Preparation of benzyl 1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate.
[00255] To a solution of 3-(benzyloxycarbonylamino)-4-cyclobutyl-2-hydroxybutanoic acid (600 mg, 1.95 mmol) in DCM (20 mL) was added HOSu (337 mg, 2.93 mmol), EDC
(562 mg, 2.93 mmol). After stirring for 1 hr, cyclopropylamine (223 mg, 3.9 mmol) was added to the above mixture. The product was extracted with EtOAc. The combined organic layer was then washed with HCl (IN), water, NaHCO3, and brine and then concentrated in vacuo to afford benzyl 1-cyclobutyl-4-(cyclopropylamino)-3-hydroxy-4-oxobutan-2-ylcarbamate (530 mg). (M+1) 347.

H2/Pd(OH) Cbz HO HN-a HO HN-a Preparation of 3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide.
[00256] To a solution of the CBz amide (530 mg, 1.53 mmol) in MeOH (30 mL) was added Pd(OH)2/C (106 mg). The mixture was stirred under H2 (1 atm) for 18 hrs. After filtration, the filtrate was concentrated in vacuo to afford the title compound (300 mg).
'H-NMR
(CDC13): S 3.29 (m, IH), 2.74 (m, 1 H), 2.37-1.66 (m, 9H), 1.40 (m, 1 H), 0.78 (m, 2H), 0.51 (m, 2H). (M+1) 213.
[00257] The following compounds were prepared in a similar fashion to preparing 3-amino-4-cyclobutyl-N-cyclopropyl-2-hydroxybutanamide by using the appropriate amine:

O ~ O O
0 ~F
H2N NHz H2N N H2N N H2N N
OH OH H OH and OH H F
Preparation of 3-amino-N-cyclopropyl-2-hydroxyhept-6-ynamide I I

HZN H
N
OH
[00258] 3-Amino-N-cyclopropyl-2-hydroxyhept-6-ynamide was prepared as described by N.
Kobayashi, et al. in US 2003/153788, which is incorporated herein by reference in its entirety. 'H-NMR (500 MHz, DMSO-d6): 8.18 (s), 6.34 (s), 4.22 (s), 3.45 (s), 3.17 (s), 2.84 (s), 2.69 (d, J = 3.2 Hz), 2.30 (m), 2.24 (m), 1.70 (m), 1.59 (m), 0.62 (d, J
= 5.0 Hz), 0.53 (s) ppm; FIA m/z 197.01 ES+.
Preparation of Cbz-protected (3S)-3-amino-4-cyclopropyl-2-hydroxy-N-methylbutanamide Cbz Na2S204 [CbNNa1 z KCN Cbz HCI
HN H o MeOH/water o C

O 2 hr HO 91%

HN O LiOH HN HOSu/EDC HN O
Cbz Cbz Cbz HO O HO OH HZN- HO HN-Step 1: Preparation of benzyl (2S)-1-cyano-3-cyclopropyl-l-hydroxypropan-2-ylcarbamate.

Cbz HN
CN
HO
[00259] To a solution of the aldehyde (7.9 g, 32 mmol) in MeOH (50 mL) at 10 C was added Na2S2O4 (6.13 g, 35.2 mmol) and the resulting mixture was warmed to room temperature and stirred for 2 hrs then cooled to 10 C. To this reaction mixture, a solution of KCN in water (50 mL) was added. After stirring at room temperature for 18 hrs, the mixture was extracted with TBME (100 mL, twice). The combined organic layers were washed with water and brine, dried and concentrated in vacuo to afford the title compound (8 g). (M+1) 275.

Step 2: Preparation of (3S)-methyl 3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoate.

HN O
Cbz /
[00260] To a solution of the cyanohydrin (1 g, 3.65 mmol) in MeOH (15 mL) at -20 C was bubbled through a stream of dry HC1 gas for 30 minutes. The resulting mixture was stirred at room temperature for 2 hrs. The reaction mixture was purged with nitrogen gas for 30 minutes and then concentrated. The residue at 0 C was quenched with ice water and then stirred at room temperature for 1 hr. The product was extracted with EtOAc.
The combined organic layer was washed with NaHCO3, water, brine and concentrated in vacuo to afford the title compound (0.5 g). 'H-NMR (CDC13) 8: 7.31-7.30 (m, 5H), 5.09 (d, 2H), 4.44-4.14 (m, 2H), 3.78 (d, 3H), 1.58-1.42 (m, 2H), 0.70 (m, 1H), 0.47 (t, 2H), 0.11-0.01 (m, 2H). (M+1) 308.
Step 3: Preparation of (3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid HN O
Cbz HO OH
[00261] To a solution of the methyl ester of Step 2 (400 mg; 1.3 mmol) in THF
(8 mL) and water (6.63 mL) was added LiOH (1 N; 1.37 mL). The reaction mixture was stirred for 30 minutes and then acidified with 1.0 N HCl to pH = 3-4. The mixture was extracted with EtOAc (20 mL, twice). The combined organic layer was washed with water, brine, and then concentrated in vacuo to afford the title compound (370 mg). (M+1) 294.
Step 4: Preparation of benzyl (2S)-1-cyclopropyl-3-hydroxy-4-(methylamino)-4-oxobutan-2-ylcarbamate.

Cbz HO HN-[00262] To a solution of (3S)-3-(benzyloxycarbonylamino)-4-cyclopropyl-2-hydroxybutanoic acid (180 mg, 0.26 mmol) in DCM (20 mL) was added HOSu (105 mg, 0.92 mmol), EDC (175 mg, 0.92 mmol). After stirred for 30 minutes, methylamine in THF
(2 N, 0.92 mL) was added to above mixture. The reaction mixture was stirred for 18 hrs and then concentrated in vacuo. The residue was purified by Gilson Prep to afford title compound (50 mg). 'H-NMR (CDC13): S 7.53-7.26 (m, 5H), 6.83 (NH, 1 H), 5.25 (NH, 1H), 5.05 (m, 2H), 4.25-3.89 (m, 3H), 2.70 (m, 3H), 1.4 (m, IH), 0.86 (m, IH), 0.61 (m, IH), 0.38 (m, 2H), 0.33 (m, 2H). (M+1) 307.
[00263] The following compounds can be prepared in the similar manner by using appropriate amines, followed by hydrogenation.

O O
H2N `NHz HzN N
OH and OH H
[00264] The following compounds can be prepared in the methods described by Perni, R. et al. in WO 01/74768, which is incorporated herein by reference in its entirety.

O O O ~ O

illlu- NHZ HzN iy - N~ HZN iyl- N H2N iyl- N
OH OH OH H , and OH H
Preparation of (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid + a O Acetone o OH
H2N COZH OJkO N sat. NaHC03 O` N
0 80% H 0 1002651 In a 5L RB flask dissolved t-butyl glycine (74 g, 0.56 mol, 1.02 eq.) in saturated sodium bicarbonate (11 vol). Cyclopentyl 2,5-dioxopyrrolidin-1-yl carbonate (126 g, 0.55 mol, 1 eq.) was dissolved in acetone (5.5 vol) and the solution slowly added via addition funnel at room temperature to the solution of the glycine. The reaction mixture was stirred at room temperature until complete (approximately 4 hrs). The acetone was removed under reduced pressure and the remaining aqueous solution was extracted with 30%
ethyl acetate in hexanes (thrice, 5.5 vol each). The organic layers were discarded.. The pH of the aqueous layer was adjusted to 2 with 2 N HC1 and then extracted with ethyl acetate (thrice, 5.5 vol).
The combined organic layers were dried (Na2SO4), filtered, and the solvent removed under reduced pressure to provide a clear oil the slowly crystallized. The crude product was crystallized from hexanes/ethyl acetate to provide (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid as a white solid (82 g). The mother liquid was stripped and a second crop of crystals obtained (combined yield 105.54 g).
Preparation of Sulfonyl Compounds i0 O O t~C-I O O ~a O O TFA
C1SN ~ i0'-p ,C ~ kM5~~ -- f~f ~IWi C1, C~ ~31V a-~p2 H
~2 H CHA H H =TFA

[00266] Compounds S1, S2, S3, and S4, shown above, were prepared according to procedures described in WO 2005/095403 and PCT/US2005/010494, hereby incorporated by references by their entireties. Specifically, to a solution of chlorosulfonylisocyanate (10 mL, 115 mmol) in CH2Cl2 (200 mL) at 0 C was added t-BuOH (11 mL, 1 eq.). The mixture was stirred for 60 minutes, then added via cannula into a solution of cyclopropylamine (6.6 g) in CH2CI2 (200 mL) with triethylamine (30 mL) at 0 C concurrently with a solution of triethylamine (50 mL) in CH2C12 (100 mL) via addition funnel. Internal temperature was maintained below 8 C. Stirred at room temperature after completion of addition for 4 hrs.
The reaction was then diluted with CH2C12 and transferred to a separatory funnel, washed with I N HC1(twice, 400 mL each), brine (300 mL), dried (MgSO4), filtered and concentrated. The product was recrystallized from ethyl acetate/hexanes to yield 16.8 g (71.3 mmol, 62 %) of S3. Compound S3 was deprotected with trifluoroacetic acid in CH2Cl2 to give compound S4 in quantitative yield.

~.0 NH3 ~.,0 CI , THF H2N1~"-7 [002671 Ammonia gas was bubbled through a gas dispersion tube into THF (40 mL) cooled to 0 C for 5 minutes. To this solution at 0 C was added cyclopropylsulfonylchloride (1 gram, 7.1 mmol). The reaction was stirred at room temperature overnight, then filtered through a plug of silica gel, followed by elution with EtOAc to yield 750 mg (6.19 mmol, 87%) of cyclopropylsulfonamide. 'H-NMR (500 MHz, Methanol-d4): 4.79 (s, 2H), 2.59-2.54 (m, 1 H), 1.06-0.96 (m, 4H).
O o CO CH CO CH P~ OMe COZCH CO H
HOZC' 2 3 OHC~- 2 3 NOMe ~ 3 ~ 2 z 0 o 0 [00268] To a solution of compound XX5 (1.37 g, 6.41 mmol) in THF (30 mL) at 0 C was added dropwise borane-dimethylsulfide (3.85 mL, 7.8 mmol, 2.0 M in toluene).
The reaction mixture was stirred for 1 h with gradual warming to room temperature, quenched with H20 (20 mL), and extracted with ethyl acetate (thrice, 30 mL each). The combined organics were dried and concentrated under reduced pressure to provide 1.3 g of a colorless oil which was used without further purification. To oxalyl chloride (2.24 mL, 25.6 mmol) in CHZC12 (15 mL, anhydrous) at -78 C under inert atmosphere was added dropwise a solution of DMSO
(2.73 mL, 38.5 mmol) in CH2C12 (8 mL). After stirring for 10 min, a solution of the alcohol (1.3 g, 6.41 mmol) in CH2C12 (6 mL) was added dropwise. After an additional 10 min, triethylamine (7.15 mL, 51.3 mmol) in CH2C12 was added and the reaction was stirred .another 30 min with gradual warming to 0 C. The reaction mixture was washed with 1 M
HCl (20 mL) followed by brine (20 mL). The organic layer was dried over MgSO4 and concentrated under reduced pressure. The resulting oil was purified via silica gel chromatography to afford 748 mg (59% over 2 steps) of aldehyde XX6. IH-NMR
(500 MHz, CDC13): 9.75 (s, 1 H), 3.67 (s, 3H), 2.91-2.85 (m, 1 H), 2.78-2.74 (m, 1 H), 2.56-2.52 (m, 1 H), 1.74-1.71 (m, 2H), 1.66-1.58 (m, 4H), 1.27-0.95 (m, 5H).
[00269] To a solution of compound XX6 (581 mg, 2.9 mmol) and K2C03 (811 mg, 5.9 mmol) in MeOH (15 mL) was added dimethyl 1-diazo-2-oxopropylphosphonate (676 mg, 3.5 mmol, Synlett 1996, p. 521). The reaction was stirred 1 h at room temperature, diluted wth Et20 (20 mL), and washed with saturated NaHCO3 solution (10 mL, aqueous). The organic layer was dried over MgSO4 and concentrated under reduced pressure to give 600 mg (100%) of alkyne XX7 which was used without further purification. 'H-NMR (500 MHz, CDC13):
3.69 (s, 3H), 2.48-2.37 (m), 1.95 (s, H), 1.73-1.60 (m), 1.30-0.94 (m).
[00270] To a solutiori of compound XX7 (600 mg, 2.9 mmol) in a solution of THF

MeOH (25 mL, 2:1:2) was added LiOH monohydrate (850 mg, 20.3 mmol). The reaction mixture was stirred 2 h at room temperature, acidified using 1 N HCl (25 mL), and extracted with EtOAc (thrice, 15 mL each). The combined organics were dried over MgSO4 and concentrated to yield 533 mg (99%) of carboxylic acid XX8, which was used without further purification.

H02C'C02CH3 N\ COZCH3 N` COZH
0j ~ 0 O
O O

[00271] To a solution of compound XX5 (100 mg, 0.5 mmol) in CH2C12 (2.5 mL) was added EDC (107 mg, 0.6 mmol), HOBt (76 mg, 0.6 mmol) and triethylamine (195 L, 1.4 mmol).
To the activated acid solution was added methylamine hydrochloride (38 mg, 0.6 mmol) and the reaction was stirred at room temperature for 12 h. The reaction mixture was washed with H20 (2 mL), 1 N HCl (2 mL) and saturated NaHCO3 solution (2 mL). The organic layer was dried over MgSO4 and concentrated to give 100 mg of amide XX9, which was used without further purification. 'H-NMR (500 MHz, CDC13) 3.61 (s, 3H), 2.75 - 2.70 (m, 4H), 2.48 -2.42 (m, 1H), 2.28 - 2.24 (m, 1H), 1.66 -1.48 (m, 6H), 1.35 - 0.90 (m, 5H).
[00272] To a solution of compound XX9 (100 mg, 0.5 mmol) in a solution of THF
/ HZO /
MeOH (3 mL, 2:1:2) was added LiOH monohydrate (124 mg, 3 mmol). The reaction mixture was stirred 2 h at room temperature, acidified using 1 N HCl (4 mL), and extracted with EtOAc (3 x 5 mL). The combined organics were dried over MgSO4 and concentrated to yield 87 mg of carboxylic acid XX10, which was used without further purification. 'H-NMR (500 MHz, CDC13) 11.32 (s, H), 2.75 - 2.64 (m, H), 2.52 - 2.46 (m, H), 2.37 - 2.33 (m, H), 2.25 (td, J = 8.7, 2.9 Hz, H), 1.97 (s, H), 1.79 (s, H), 1.74 - 1.62 (m, H), 1.59 -1.49 (m, H), 1.23 -1.12 (m,.H), 1.08 - 0.81 (m, H).

HO2C'-'-,CO2CH3 0CO2CH3 10 0CO2H

0a O 0 O
XX5 XXl l XX12 [00273] Intermediate XX12 was prepared according to the procedure for preparing intermediate XX10 described above, except for using pyrrolidine as a reagent instead of methylamine hydrochloride. IH-NMR (500 MHz, CDC13) 11.47 (s, IH), 3.45 - 3.32 (m, 4H), 2.76 - 2.72 (m, IH), 2.64 - 2.59 (m, 1 H), 2.37 - 2.33 (m, IH), 1.92 - 1.76 (m, 4H), 1.71 - 1.57 (m), 1.22 - 0.84 (m).

~COzCH3 Br~,CO2CH3 ~iCOZCH3 ~iCO2H

O -> -~ -- ' (D 0 [00274] To a solution of compound XX5 (1 g, 4.7 mmol) and HgO yellow (1.01 g, 4.7 mmol) in CC14 (23 mL) at reflux was added dropwise over 30 min a solution of bromine (264 L, 5.1 mmol) in CC14 (5 mL). The reaction was stirred at reflux for 1 h, cooled to room temperature, diluted with CH2C12 (20 mL), washed with 1 N HCl (10 mL), H20 (10 mL), and brine (10 mL). The organic layer was dried over MgSO4 and concentrated under reduced pressure to yield 1.3 g of compound XX13 as a colorless oil that was used without further purification. 1H-NMR (500 MHz, CDC13): 3.67 (s, 3H), 3.52 - 3.44 (m, 2H), 2.63 - 2.58 (m, 1H), 1.70 - 1.64 (m, 3H), 1.60 - 1.54 (m, 3H), 1.24 - 0.92 (m, 5H).
[00275] To a solution of compound XX13 (578 mg, 2.3 mmol) in DMSO (12 mL) was added sodium borohydride (177 mg, 4.7 mmol). The reaction mixture was stirred at 90 C
for 1 h, diluted with H20 (10 mL), and extracted with hexanes (3 x 15 mL). The combined organics were dried over MgSO4 and concentrated under reduced pressure.
Purification via silica gel chromatography, eluting with EtOAc / petroleum ether, afforded 204 mg of compound XX14. 1H-NMR (500 MHz, CDC13): 3.59 (s, 3H), 2.18 (m, 1H), 1.69 -1.43 (m, 6H), 1.21 - 0.83 (m, 8H).
[00276] Intermediate XX15 was prepared according to the procedure for preparing intermediate XX10, step b, except for using substrate XX14 instead of XX9.

Br H COzH

[00277] To a solution of (S)-2-amino-3,3-dimethylbutanoic acid (787 mg, 6.0 mmol), bromobenzene (632 L, 6.0 mmol), K2C03 (1.24 g, 9.0 mmol) and CuI (114 mg, 0.6 mmol) was added N,N-dimethylacetamide (7.5 mL). The contents were stirred for 16 h at 90 C in a sealed pressure vessel. The reaction mixture was diluted with H20 (15 mL), cooled to 0 C, and acidified to pH - 5 using 1 N HC1. The mixture was extracted with EtOAc (3 x 20 mL), and the combined organics were washed with brine (1 x 15 mL), dried over MgSO4, and concentrated under reduced pressure. The resulting residue was purified via silica gel chromatography to provide 150 mg (12%) of compound XX16. 1H-NMR (500 MHz, CDC13): 7.11-7.09 (m, 2H), 6.69 (t, J = 7.3 Hz, 1 H), 6.60- 6.59 (m, 2H), 3.69 (s, 1 H), 1.02 (s, 9H).

MeO H N CO2H

[00278] Intermediate XX17 was prepared according to the procedure for preparing XX16, except for using 1-bromo-3-methoxybenzene as a reagent instead of bromobenzene. I H-NMR (500 MHz, CDC13): 6.98 (t, J = 8.1 Hz, 1 H), 6.24-6.18 (m, 2H), 6.14 (s, 1 H), 3.69 (s, 1H), 3.66 (s, 3H), 1.00 (s, 9H).

a,,,,,~C02CH3 HO2C'COZCH3 NN~COZH
O ~ 0 [00279] To a solution of (S)-3-(methoxycarbonyl)-4-methylpentanoic acid (200 mg, 1.2 mmol) in CHZCIz (6 mL) was added EDC (264 mg, 1.4 mmol), HOBt (186 mg, 1.4 mmol) and triethylamine (481 L, 3.5 mmol). To the activated acid solution was added cyclohexylamine (158 L, 1.4 mmol) and the reaction was stirred 4 hrs. The reaction mixture was washed with H20 (3 mL), 1 N HCl (3 mL), and saturated NaHCO3 solution (3 mL). The organic layer was dried over MgSO4, and concentrated under reduced pressure to afford 290 mg of compound XX18 which was used without further purification. I H-NMR (500 MHz, CDC13): 5.78 (d, J=7.5 Hz, 1H), 3.69-3.61 (m, 4H), 2.73-2.69 (m, 1H), 2.45-2.40 (m, 1 H), 2.24-2.20 (m, -1H), 1.85 (m, 1H), 1.82-1.76 (m, 2H), 1.63-1.60 (m, 2H), 1.54-1.50 (m, 1H), 1.31-1.22 (m, 2H), 1.12-1.00 (m, 3H), 0.90-0.85 (m, 6H).
[00280] Intermediate XX19 was prepared according to the procedure for preparing compound XX10 described above, except for using substrate XX18 as a reagent instead of compound XX9. ES (+) MS: m/e 256 (M + H)+.
H
N\^/C02H
Y
j0 ( [00281] Intermediate XX20 was prepared according to the procedure for preparing compound XX18 or XX19 described above, except for using isopropylamine as a reagent instead of cyclohexylamine. ES (+) MS: m/e 216 (M + H)+.

H
N\^/C02H
0( 1002821 Intermediate XX21 was prepared according to the procedure for preparing XX18 or XX19 described above, except for using benzylamine as a reagent instead of cyclohexylamine. ES (+) MS: m/e 264 (M + H)+.

O + HCI = H2NCH2CO2CH3 Ul"-" N CO CH
~/ 2 3 1002831 Glycine methyl ester hydrochloride (50.0 g) was suspended in MTBE (300 mL) at RT. To this was added benzaldehyde (40.5 mL) and anhydrous Na2SO4 (33.9 g).
The suspension was cooled in an ice-water bath for 20 minutes, then triethylamine (80 mL) was added dropwise over 15 minutes. After 5 minutes, the reaction was removed from the ice-water bath, and stirred at RT for 24 hrs. The reaction was quenched with 200 mL ice-water mixture and the organic layer was separated. The aqueous layer was extracted with MTBE
(200 mL). The organic layers were combined, washed with a 1:1 mixture of brine and saturated NaHCO3 (aq.), dried (MgSO4), and concentrated to yield 62.83 grams of the N-benzyl imine as a yellow oil. 'H-NMR (500 MHz, CDC13): 8.30 (s, IH), 7.78-7.77 (m, 2H), 7.45-7.40 (m, 3H), 4.42 (s, 2H), 3.78 (s, 3H).

H O
, N,, Oi I Boc - ( ) [00284] _Lithiumtert=butoxide (15.13 g) was suspended in dry toluene (200 mL) at room remperature. To this was added dropwise a solution of the N-benzyl imine of glycine methyl ester (16.89 g) and 1,4-dibromo-2-butene (19.28 g) in toluene (100 mL) over 40 minutes.
The red solution was stirred for 100 minutes, then quenched with H20 (200 mL).
The contents were transferred to a separatory funnel and diluted with MTBE (200 mL). The layers were separated and the aqueous layer was extracted with MTBE. The combined organic layers were stirred with 1 N HC1(aq.) (500 mL) for 3 hrs. The layers were separated and the organic layer was extracted with H20 (100 mL). The aqueous layers were combined, NaC1(250 g) and MTBE (700 mL) were added and the pH was brought to - 13 with NaOH (aq). The organic layer was separated and the aqueous layer was extracted with MTBE (twice, 300 mL each). The organic layers were combined, dried (MgSO4), and concentrated to a volume of z 400 mL. To the solution was added di-tert-butyl dicarbonate (25.0 g) and the reaction was stirred for 3 days. Additional di-tert-butyl dicarbonate (5.6 g) was added, followed by heating of the reaction in a 60 C bath for 1 hr. The reaction was purified by flash silica gel column chromatography with EtOAc/hexane (1:9) as eluent to yield 10.89 g of racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester. See, e.g., W000/09558 and Beaulieu, P. L.et al., J. Org.
Chem., 70 (15), 5869 -5879, 2005. 'H-NMR (500 MHz, CDC13): 5.78-5.71 (m, IH), 5.29-5.26 (m, IH), 5.11 (dd, J=1.2, 10.3 Hz, 1H), 3.71 (s, 3H), 2.14 (q, J=8.8 Hz, 1H), 1.79 (s, 1H), 1.53-1.45 (m, l OH).

H O H O
Boc' N'~ O~ Boc' N'~R O
(S) ( ) 1002851 Racemic N-Boc-(1 R,2S)/(1 S,2R)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester (4.2 g) was dissolved in acetone (80 mL) and then diluted with water (160 mL).
The pH was adjusted to 7.8 with 0.2N NaOH (aq). Subtilisin A (product P-5380 from Sigma, St. Louis, MO, USA) (4.5 g) was added to the solution. Its pH was maintained between 7.4 and 8.7 for 3 days by the dropwise addition of 0.1 N NaOH (aq.). When HPLC
analysis (Chiralpak AD from Daicel Chemical Industries, Tokyo, 4.6 mm x 250 mm, 0.5 mL/min, 10-85% 2-propanol/hexanes over 10 minutes, monitor 215.4 nm) of the reaction indicated the presence of only the (1R,2S)-enantiomer (retention time of (1R,2S) = 6.2 min, (1S,2R) = 5.9 min) the pH was brought to 8.5 with 2 N NaOH (aq). The contents of the reaction were -transferred to a separatory funnel and extracted with MTBE (3 X 400 mL). The extracts were washed with saturated NaHCO3 (aq) solution (3 X 150 mL), water (2 X 200 mL), and dried (MgSO4). The solution was filtered, concentrated, diluted with CH2C12, dried (MgSO4), filtered, and concentrated to yield 1.95 g ofN-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester.
H O O
Boc' N'~R Oi TFA - H2N,(R O
(S) ON
(S
[00286] N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester (125 mg, 0.52 mmol) stirred in CH2C12/TFA (1:1, 2 mL) at RT for 90 minutes. Solvents removed under vacuum to yield (1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid methyl ester trifluoroacetic acid salt.

H O H O
Boc' Nl. O Fmoc' N/. OH

XXl XX2 [00287] Compound XX1 (2.34g, 9.71 mmol) was stirred with LiOH=H2O (0.45 g, 10.7 mmol) in THF/H20/THF (3:1:0.5, 22 mL) at room temperature overnight. The solvents were evaporated and the remaining solids were taken up in CH2C12/EtOAc and 1 N HCl (aq). The aqueous layer was extracted with CH2C12 and the combined organic extracts were dried (MgSO4), filtered, and concentrated. This material was dissolved in CH2C12 (10 mL) at room temperature and treated with trifluoroacetic acid (10 mL). HPLC analysis at 70 minutes showed no starting material was present. The solvents were removed in vacuo to yield a viscous light colored oil. This was taken up in additional CH2C12 (30 mL) and evaporated on a rotary evaporator to yield a tan solid. This solid was dissolved in saturated NaHCO3 (aq) and acetone (1:1, 50 mL) and treated with Fmoc-Cl (2.65 g, 10.2 mmol). After 4 hrs, the contents of the flask were transferred to a separatory funnel with CH2C12 and acidified with 2N HCl (aq). The aqueous layer was extracted with CHzCIZ, the combined organic layers were dried (MgSO4), filtered, and concentrated to yield 1.86 g (5.3mmol) of XX2 as a light yellow solid. (M+1) = 350.1 PS-Wang Resin o DIC, DMAP 0 c o N DMF, DCM o~N oy OH

[00288] PS-Wang resin (2.0g, l.0eq.) swelled in DMF (enough to cover).
(IR,2S')-1-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-vinylcyclopropanecarboxylic acid (XX3) (922 mg, 1.1 eq.) was stirred in DCM. Diisopropylcarbodiimide (409uL, 1.1 eq.) was added to the DCM solution and stirred at 4 C for 2 hrs, then added to resin and DMF.
Dimethylaminopyridine (29mg, 0.1eq.) in DMF was added to resin solution and shaken for 5 hrs. Drained and washed with DMF (thrice) and DCM (thrice) to yield Compound XX4.

DiOEt 0~'iOEt ~OH

Xi X2 Preparation of 2-(bicyclo[4.1.0]heptan-l-yl)acetic acid X2:
1002891 Commercially available compound Xl (Aldrich Chemical Co., Milwaukee, Wisconsin, USA) was converted to X2 according to method described by E. J.
Kantorowski et al. in J. Org Chem., 1999, 64, 570-580. 1 H-NMR (CDC13, 500 MHz): 9.2 (br s, 1 H), 2.23 (m, 2H), 1.92 (m, 1 H), 1.76 (m, 2H), 1.58 (m, 1 H), 1.34 (m, IH), 1.18 (m, 4H), 0.85 (m, 1 H), 0.52 (dd, 1 H), 0.31 (t, 1 H) ppm.
O

~OH
OH OH

Preparation of 2-(1-hydroxycyclohexyl)acetic acid X5:
[002901 Compound X4 was prepared using essentially the procedure described in Bull.
Chem. Soc. Jpn., 1971, 44, 1090. Specifically, A solution of ethylbromoacetate (8.3 mL) (Aldrich Chemical Co., Milwaukee, Wisconsin, USA) in toluene was added dropewise at 80 C over 30 min. to a thoroughly stirred mixture of cyclohexanone X3 (4.9 g) and zinc powder (4.9 g) in toluene. The addition was carefully monitored and the temperature was kept at 80 C. After the addition was completed, the mixture was refluxed for 90 min., cooled, decomposed with 1N aqueous HCI, and extracted with Et20. The organics were washed with water, aq. NaHCO3, dried (MgSO4) and concentrated in vacuo to yield X4 (5.9g): tH-NMR (CDC13, 500MHz) 4.16 (t, 2H), 3.0 (br s, 1H), 2.46 (s, 2H), 1.40-1.69 (m, 10H), 1.27 (t, 3H) ppm; FIA m/z 187.1 ES+.
[002911 To a solution of X4 (510 mg) in MeOH was added 1N aqueous NaOH. The reaction mixture was stirred at 60 C for 1 h, and then concentrated in vacuo. The residue was diluted with water, washed with Et20 and the aqueous layer acidified with 1N aqueous citric acid and extracted with EtOAc. The organics were dried (MgSO4) and concentrated in vacuo to yield after recrystallization compound X5 (220mg): 1H-NMR (CDC13, 500MHz) 3.63 (s, 1H), 2.45 (s, 2H), 1.22-1.64 (m, lOH) ppm; FIA m/z 157.2 ES-.

~ -~ OEt OEt OH

Preparation of 2-(1-methylcyclohexyl)acetic acid (X8) [00292] Commercially available compound X6 (Aldrich Chemical Co., Milwaukee, Wisconsin, USA) was converted to compound X7 according to the method described by N.
Asao et al. in Tetrahedron Lett., 2003, 44, 4265. 'H-NMR (CDC13, 500 MHz):
4.12 (q, 2H), 2.22 (s, 2H), 1.30-1.48 (m, lOH), 1.25 (t, 3H), 1.01 (s, 3H) ppm.
'[00293] To a solution of compound X7 in EtOH was added 1 N aqueous NaOH. The reaction mixture was stirred at 50 C for 3 hrs, and then concentrated in vacuo. The residue was diluted with water, washed with Et20 and the aqueous layer acidified with 1 N aqueous citric acid and extracted with CH2C12. The organics were dried (MgSO4) and concentrated in vacuo to yield compound X8. 'H-NMR (CDC13, 500MHz): 11.7 (s, 1H), 2.26 (s, 2H), 1.32-1.49 (m, 10H), 1.05 (s, 3H) ppm.

o o o~-l O o'1 0 O ~ OEt ~OEt -~OH

X9 X10 Xil X12 Preparation of 2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid (X12) [00294] To a solution of dihydro-2H-pyran-4(3H)-one (X9) (3.13 g, from Aldrich) in toluene was added (carbethoxymethylene)-triphenylphosphorane (12.0 g, Aldrich). The solution was stirred at 110 C for 3 days. The resulting dark solution was concentrated in vacuo and the residue directly purified by column over silica gel to yield compound X10 (4.54 g) as a clear liquid. 'H-NMR (CDC13, 500MHz): 5.66 (s, 1H), 4.16 (q, 2H), 3.98 (s, 4H), 3.00 (t, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.
[00295] Compounds Xl l and X12 were obtained in a similar manner as described for compounds X7 and X8. 'H-NMR (CDC13, 500 MHz): 3.64-3.73 (m, 4H), 2.35 (s, 2H), 1.65 (ddd, 2H), 1.50 (ddt, 2H), 1.17 (s, 3H) ppm.

0 ~
I Q" 1 0 0 0 0 0 ~~O OEt OEt OH
O

Preparation of 2-(cis-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid (X16) [00296] Intermediate X13 was prepared from commercially available 2,6-dimethyl-g-pyrone (Aldrich Chemical Co., Milwaukee, Wisconsin, USA). A solution of the g-pyrone was dissolved in EtOH and hydrogenated (2 atm. H2) with 10% Pd/C over 2h. The catalyst was subsequently filtered off and the solution was concentrated in vacuo to yield crude X13 which was purified by column chromatography to yield pure compound X13. 'H-NMR
(CDC13, 500 MHz): 3.72 (m, 2H), 2.35 (m, 2H), 2.21 (dd, 2H), 1.32 (d, 6H) ppm.
[00297] Compound X14 was then obtained from compound X13 in a similar manner as described for compound X10. 'H-NMR (CDC13, 500 MHz): 5.65 (s, 1H), 4.15 (q, 2H), 3.80 (dt, 1 H), 3.49 (m, 2H), 2.17 (dt, 1 H), 2.07 (dd, 1 H), 1.79 (dt, 1 H), 1.28 (m, 9H) ppm. LC-MS
m/z 199.126 ES+.
[00298] A solution of compound X14 in EtOAc was then hydrogenated (1 atm. H2) with 10% wet Pd/C over 1 hr. The catalyst was subsequently filtered off and the solution was concentrated in vacuo to yield crude compound X15 which was used without further purification for the next step. Compound X16 was then prepared from compound X15 in a similar manner as described for compound X8. tH-NMR (CDCl3i 500 MHz) major diastereomer: 3.50 (m, 2H), 2.27 (d, 2H), 2.07 (m, 1H), 1.71 (m, 2H), 1.19 (d, 6H) 0.92 (m, 2H) ppm; major diastereomer: 3.64 (m, 2H), 2.56 (d, 2H), 2.47 (m, 1H), 1.49 (m, 2H), 1.15 (d, 6H), 0.86 (m, 2H) ppm.
~~ ---~ 00 , ---~ ~-~~ ~~
p OEt OEt OH

Preparation of 2-(1,4-dioxaspiro[4.5]decan-8-yl)acetic acid X20:
[00299] Compound X20 was prepared from compound X17 (from Aldrich) according to the procedures described above for preparing compound X16.
[00300] Compound X18: 'H-NMR (CDC13, 500 MHz): 5.66 (s, 1H), 4.15 (q, 2H), 3.98 (s, 4H), 3.00 (m, 2H), 2.38 (m, 2H), 1.77 (m, 4H), 1.27 (t, 3H) ppm.
[00301] Compound X19: I H-NMR (CDC13, 500 MHz): 4.12 (q, 2H), 3.93 (s, 4H), (d, 2H), 1.83 (m, 1H), 1.72 (m, 4H), 1.56 (dt, 2H), 1.33 (m, 2H), 1.30 (m, 3H) ppm.
[00302] Compound X20: 'H-NMR (CDC13, 500 MHz): 3.93 (s, 4H), 2.28 (d, 2H), 1.73-1.86 (m, 4H), 1.57 (dt, 2H), 1.35 (m, 2H) ppm.
i~OMe O
TMSO O+ r Y "~ O O O OEt O O OEt O O OH IIL AH 0 I I

Preparation of.2-(trans-2,6-dimethyltetrahydro-2H-pyran-4-yl)acetic acid 25:
[00303] Compounds X21 and X22 were prepared according to the method described by S.
Danishefsky et al. in J. Org. Chem. 1982, 47, 1597-1598 and D. S. Reddy et al.
in J. Org.
Chem. 2004, 69, 1716-1719, respectively. Compound X25 was prepared from compound X22 according to the method described above for preparing compound X16.
[00304] Comopund X23. 'H-NMR (CDC13, 500 MHz): 5.72 (s, 1H), 4.16 (q, 2H), 4.08 (q, 2H), 3.06 (dd, 1 H), 2.75 (dd, 1 H), 2.39 (dd, IH), 2.05 (dd, 1 H), 1.28 (t, 3H), 1.19 (m, 6H) ppm.
[00305] X25: 'H-NMR (CDC13, 500 MHz) 4.24 (m, 1H), 3.78 (m, 1H), 2.25 (m, 3H), 1.71 (m, 1 H), 1.53 (m, 1 H), 1.46 (m, 1 H), 1.29 (d, 3H), 1.13 (d, 3H), 0.90 (m, 1 H) ppm.

~ ~ ~~
OH OH OH

Preparation of 2-(4-hydroxy-4-methylcyclohexyl)acetic acid X27:

1003061 A solution of compound X20 in dioxane was treated with 4N HCl in dioxane. The reaction solution was stirred at room temperature for 4 hrs and concentrated in vacuo to give crude compound X26 which was used without further purification for the next step. To a stirred solution of compound X26 in THF was slowly added MeMgBr (3 N in THF).
The resulting mixture was stirred at 40 C for 3 hrs, quenched with 1 N aqueous citric acid and diluted with EtOAc. The phase were separated and the organics were dried (MgSO4), concentrated in vacuo and purified by chromatography over silica gel to give compound X27 as a mixture of two diastereomers: isomer 1: 'H-NMR (CDC13, 500 MHz): 4.50 (br s), 2.27 (m, 2H), 1.75 (m, 1H), 1.65 (m, 4H), 1.39 (m, 4H), 1.22 (s, 3H) ppm; isomer 2:
'H-NMR
(CDC13, 500 MHz): 2.12 (m, 2H), 1.69 (m, 3H), 1.56 (m, 2H), 1.39 (m, 2H), 1.12 (s, 3H), 1.05 (m, 2H) ppm.

O LiOH O O
O~ OH
Preparation of 2-(2,2-dimethyltetrahydro-2H-pyran-4-yl)acetic acid [00307] To a solution of the methyl ester (500 mg; 2.69 mmol) in THF (21.5 mL), MeOH
(21.5 mL) and water (10.75 mL) was added LiOH (1 N; 10.75 mL). The reaction mixture was stirred for 3 hrs. The reaction was acidified with HCl (1 N, pH = 5). The product was extracted with EtOAc (twice, 20 mL each). The combined organic layer was then wash with water, brine and concentrated in vacuo to afford 420 mg of 2-(2,2-dimethyltetrahydro-2H-pyrari=4-y1)acetic acid. 'H-NMR (CDC13): S 3.76-3.67 (m, 2H), 2.56-2.19 (m, 3H), 1.63 (m, 2H), 1.26-1.10 (m, 8H). (M+1) 173.

OH H O H
AIIocHN~N~ AIIocHN\ ~ /N~

N O~ I HN 'If, O N O~ I HN y O
O HN,NH2 O HN,N H
AIIocHN, ~ ,N,, O

[00308] To a solution of compound X30 (64 g, 237 mmol) and EDC (226 g, 1.19 mol) in EtOAc (1.5 L) was added DMSO (400 mL), and the resulting suspension was cooled to 0 C.

To this mixture was added a solution of dichloroacetic acid in EtOAc (1:1 v/v, 130 mL) keeping the internal reaction temperature below 25 C. The reaction was warmed to room temperature, stirred for 15 minutes, cooled to 0 C, and quenched with 1 N HCl (1 L). The organic layer was separated, washed with H20 (2 x 500 mL), dried over MgSO4, and concentrated under reduced pressure. The resulting oil was filtered through a plug of silica eluting with EtOAc / hexanes to afford 48 g (76%) of compound X31 as a white solid.
[00309] To resin X32 (prepared according to the procedure described in WO
00/2342 1) (100 g, 0.88 mmol/g) was added a solution of X31 (48 g, 179 mmol) in THF (650 mL), followed by AcOH (30 mL). The mixture was shaken for 16 hrs, and the resin was filtered, washed with THF (4 times, 400 mL each) and CH2CI2 (4 times, 400 mL each) and dried in vacuo.
The filtrate and washes were combined and concentrated, and the above procedure was repeated to afford resin X33 with a loading of approximately 0.4 mmol/g.
Preparation of Aldehyde Compounds [00310] 5-chloronicotinaldehyde was prepared according to methods described by D.L.
Comins et al. in Hetereocycles, 1987, 26 (8), pp. 2159-2164.
[00311] Some other aldehydes such as 2-fluoro-5-chlorobenzaldehyde, 2-methoxy-3-methyl benzaldehyde, 2-methoxynicotinaldehyde, 2,3-dihydrobenofuran-7-carbaldehyde can be made from corresponding acid based on following procedure:
1) MeOCOCI
TEA, F
2) N BH4 ~
O ~ / PCC, DCM O O

HO O HO O
Preparation of 2,3-dihydrobenzofuran-7-carbaldehyde [00312] 2,3-Dihydrobenzofuran-7-carboxylic acid (820 mg, 5 mmol) was dissolved in THF
(10 mL). To the solution was added TEA (0.7 mL, 5 mmol) and methylchloroformate (0.43 mL, 5 mmol). The solution was stirred for 0.5 hr. The white precipitates were removed by filtration, the filtrate was added to a solution of NaBH4 (437 mg, 12.5 mmol) in H20 (5 mL).
The resulting solution was stirred overnight. The reaction mixture was neutralized with 2 M
aqueous HCl solution and then extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude alcohol was -dissolved in DGM. To the solution was added PCC (1.83 g, 7.5 mmol). The mixture was stirred for 2 hrs at room temperature and diluted with diethyl ether, then ether layers were decanted. Combined organic layer was filtered though a layer of Celite . The filtrate was concentrated to give crude product. The crude was purified from column with 10%

EtOAc/hexane to afford 450 mg of 2,3-dihydrobenzofuran-7-carbaldehyde as a slightly yellow solid. HPLC 4.3 min.

CI CI
Mn02, CHCI3 ~
~ \ 1 O
~ O I-{
N
Preparation of 4-chloropicolinaldehyde [00313] A suspension of Mn02 (7.3 g, 84 mmol) and (4-chloro-pyrindin-2-yl)methanol (1 g, 7 mmol) in CHC13 was heated to refulx for 90 minutes. The mixture was filtered though a layer of Celite and concentrated in vacuo to afford 520 mg of 4-chloropicolinaldehyde as a white solid. HPLC 1.8 minutes and MS 142 as M=1 peak.
CI CI

Preparation of 3-chloro-5-methoxybenzaldehyde [00314] A mixture of 3-chloro-5-methoxybenzyl alcohol (5.0g, 28.9 mmol) and pyridinium chlorochromate (20% on alumina, 40g, 37.8 mmol) was allowed to stir for 1.25 hr. Diethyl ether. (200m1) was then added followed by filtration of precipitate. The filtrate was concentrated under reduced pressure and the resulting residue was purified via silica gel chromatography using 40% dichloromethane, 60% petroleum ether as eluant, to give 3.8g of 3-chloro-5-methoxybenzaldehyde (78%). 'H-NMR (CDC13): 3.84 (s, 3H) 7.13 (s, 1H), 7.28 (s, 1 H), 7.41 (s, 1 H), 9.89 (s, 1 H).
CI CI

Br Preparation of 1-(bromomethyl)-3-chloro-5-methylbenzene [00315] To a solution of m-chloroxylene (0.96g, 6.8mmol) in carbon tetrachloride at reflux was added N-bromosuccinmide (1.4g, 7.5 mmol) followed by benzoyl peroxide (1.6g, 6.8 mmol). The reaction was allowed to stir for 20 minutes and cooled to room temperature, filtered off precipitate and the filtrate was concentrated under reduced pressure and the resulting residue was purified via silica gel chromatography using petroleum ether as eluant to give 0.89g of 1-(bromomethyl)-3-chloro-5-methylbenzene (60%). NMR (CDC13):
2.31 (s,3H) 4.37 (s,2H) 7.09 (s,1 H) 7.12 (s,1 H) 7.20 (s,1 H).

CI Ci gr H O
Preparation of 3-chloro-5-methylbenzaldehyde [00316] To a solution of sodium metal ( 52 mg, 2.3mmo1) in ethanol was added 2-nitropropane (0.23g, 2.4 mmole) followed by the addition of 3-chloro-5-methybenzylbromide (0.5g, 2.3 mmol). The reaction was allowed to stir for 3 hrs and the precipitate formed was filtered off. The filtrate was concentrated under reduced pressure, redissolved in diethylether and washed with 1N sodium hydroxide (twice), water, and dried over sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The resulting residue was purified via silica gel chromatography using 10% dichloromethane and 90% petroleum ether, to give 0.15g of 3-chloro-5-methylbenzaldehyde (42%). 'H-NMR (CDC13): 2.46 (s, 3H) 7.43(s, 1H) 7.56 (s, 1 H) 7.68(s, 1 H), 9.92 (s, 1 H).

F F I
OH KOH O
H 6 CI H20/THF H ~ CI
O iodoethane O
[00317] '3-Cliloro-5=fluoro-4-hydroxybenzaldehyde (1.0 gram, 5.7 mmol) in THF
(40mL) was heated at reflux for 17 hrs with KOH (534 mg, 9.5 mmol, 1.7 eq) in water (5 mL) and iodoethane (1 mL, 2.2 eq). The reaction was then transferred to a separatory funnel with water and extracted with methylene chloride (thrice, 150 mL each). The combined organic layers were washed with 10 % aqueous HCl (40 mL), dried (MgSO4), and concentrated to a viscous orange liquid to yield 1.13 g of 3-chloro-4-ethoxy-5-flurobenzaldehyde (98%). 'H-NMR (500 MHz, CDC13): 9.84 (d, J=1.9 Hz, 1H), 7.71 (t, J=1.6 Hz, 1H), 7.53 (dd, J=1.9, 10.7 Hz, 1H), 4.37-4.32 (m, 2H), 1.47-1.40 (m, 3H).

I OH KOH ~

iodoethane WO 2008/106058 ' , PCT/US2008/002395 [00318] 4-Ethoxy-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 3-chloro-4-ethoxy-5-flurobenzaldehyde. 'H-NMR (300 MHz, CDC13): 9.89 (s, 1H), 7.56 (s, 2H), 3.91 (q, 7 Hz, 1H), 2.34 (s, 6H), 1.44 (t, J=7 Hz, 6H).

I OH KOH O

O 2-iodopropane O
[00319] 4-Isopropoxy-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 4-Ethoxy-3,5-dimethylbenzaldehyde. 'H-NMR (300 MHz, CDC13): 9.88 (s, 1H), 7.55 (s, 2H), 4.31 (q, J=6 Hz, 1 H), 2.32 (s, 6H), 1.32 (d, J=6 Hz, 6H).

Ac H K OH O
H2O/THF H O yclopropylmethylbromide O
'[003201 4-(Cyclopropylmethoxy)-3,5-dimethylbenzaldehyde was prepared in a manner similar to that of 4-Ethoxy-3,5-dimethylbenzaldehyde. I H-NMR (300 MHz, CDC13): 9.87 (s, 1H), 7.55 (s, 2H), 3.69 (d, J=7 Hz, 2H), 2.35 (s, 6H), 1.35-1.23 (m, 1H), 0.67-.060 (m, 2H), 0.35-0.30 (m, 2H).

Preparation of (S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid.
HO, O
TEMPO(O.05 eq) ~OH OH
NaOCi (1.1 eq) N
Boc O 0 C, 1 hr N
Boc O

[00321] A solution of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1.0 eq.) in isopropyl acetate (5 vol) was cooled to 0 C and TEMPO (0.05 eq.) was added. A solution of bleach (12.5 wt %, 1.2 eq., 2.6 vol) was then slowly added over 1 hr while maintaining the temperature at 0-5 C. The mixture was stirred and monitored by HPLC for completion, then aqueous 10% KHSO4 (2.5 vol) was added, stirred for 10 minutes, and then the phases were separated. The organic phase was washed with aqueous 5%
Na2SO3 (2 vol) then brine (1 vol) then dried azeotropically and concentrated to afford the title compound as a solid. The solid was triturated with acetonitrile (1.0 vol) to remove residual color and impurities. 'H-NMR (400 MHz, DMSO): S 4.54 (m, 1 H), 3.82 (m, 1 H), 3.67 (m, 1H); 3.15 (m, 1H); z 2.50 (m, 1H, coincides with DMSO); 1.42 and 1.39 (2 s rotamers, 9H).
Preparation of (S)-1-(tert-butoxycarbonyl)-4-methylenepyrrolidine-2-carboxylic acid Me3PBr (3.0 eq) 0 K+ t-BuO- (3.0 eq) N N
Boc 0 Boc 0 1003221 To a suspension of inethyltriphenylphosphonium bromide (2.2 eq.) in 2-methyl tetrahydrofuran (3 vol) was added rapidly solid potassium tert-butoxide (2.3 eq.) maintaining the temperature around 0 C. The temperature was kept at +20 C for 2 hrs (a suspension remained) and re-cooled to 0 C. Keeping the temperature below 6 C, (S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid (1 eq.) was added over 40 minutes. The reaction was warmed to room temperature and stirred for 16 h and then cooled to 0 C. The reaction was quenched with saturated NaHCO3 (5 vol) and water (2 vol) and the aqueous layer was separated. The organic layer was extracted with saturated NaHCO3/water (1.8 vol/1.8 vol) and the combined aqueous layers were filtered through Celite .
The aqueous layer was acidified with 6 N HCl (2.6 vol) at ambient temperature and extracted twice with isopropyl acetate (16 vol, then 8 vol). The organic phase was dried (MgSO4) and the solvent removed. -The crude product was dissolved iri*isopropyl acetate (10 vol) and extracted with 0.5 M NaOH (10 vol, then 1 vol). The combined aqueous layers were acidified at ambient temperature with 6 N HCl to pH = 3, and extracted twice with ethyl acetate (10 vol, then 8 vol). The combined extracts were dried (Na2SO4), the solvent removed and the crude product was recrystallized from cyclohexane (5 vol) to afford the title compound. 'H-NMR (400 MHz, DMSO): S 12.9, (broad, IH); 5.00 (m, 2H); 4.24 (dt, J=1.9 H, J=7.3 Hz, IH), 3.91 (m, 2H); 2.98 (m, 1H); ,z 2.50 (m, 1H, coincides with DMSO); 1.41 and 1.36 (2 s rotamers, 9H).
[00323] B. Synthesis of Exemplary Compounds of Formula I
[00324] Certain exemplary compounds of Formula I may be prepared by Method 1 as illustrated below.
[00325] All of the following reactions were performed under nitrogen and used anhydrous solvents unless otherwise noted.
Example 1: (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspirof 4.41nonane-8-carboxamide (Cmpd No. 8) [00326] Step A: (2S)-di-tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-hydroxypyrrolidine-1,2-dicarboxylate (Al) OEt ~O
OH
BoCN

O
O

Al [00327] Zinc dust (1.05 g, 16 mmol, 1.6 eq.) in a flask was heated with a heat gun two times.
Dry THF (10 mL) and chlorotrimethylsilane (0.405 mL, 3.2 mmol, 0.32 eq.) were added to this flask. The suspension was stirred for 15 min at room temp., heated to reflux, and then removed from the heat. (S)-di-tert-butyl 4-oxopyrrolidine- 1,2-dicarboxylate (2.85 g, 10 mmol, 1 eq.) and ethyl 2-bromoacetate (1.77 mL, 16 mmol, 1.6 eq.) were combined with dry THF (10 mL), and added slowly to the Zinc suspension at a rate that at which a gentle reflux was observed. The reaction was refluxed for 2 hrs and monitored by TLC. Upon completion, the reaction was concentrated. Chromatography using hexanes and ethyl acetate gave 2.64 g (7.3 mmol) of (2S)-di-tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-hydroxypyrrolidine-1,2-dicarboxylate (Al) as a 4:1 (2S,4S:2S,4R) mixture of the diastereomers. 'H
NMR (300 MHz, DMSO) 4.97 (m, 1H), 4.14 - 4.01 (m, 3H), 3.48 - 3.33 (m, 2H), 2.56 (m, 2H), 2.35 -2.19 (m, 1H), 2.03 (dd, 1 H), 1.40 - 1.35 (m, 18H), 1.18 (t, 3H).
[00328] Step B: (8S)-di-tert-butyl2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (B1) H
N
X= O
BocN O

O
O

[00329] (2S)-di-tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-hydroxypyrrolidine-1,2-dicarboxylate (Al) (1.58 g, 4.42 mmol, 1 eq.) was diluted in methanol (6 mL). To this mixture, hydrazine (0.417 mL, 13.26 mmol, 3 eq.) was added. The reaction was stirred at room temp. overnight then concentrated. To the residue, 0.5 N HCl (22.1 mL, 11.05 mmol, 2.5 eq.) was added and the mixture was sonnicated until the residue dissolved in the acid. The solution was cooled in an icewater bath. A 1.0 M solution of NaNOz (5.3 mL, 5.3 mmol, 1.2 eq.) was added slowly and stirred for 15 min. A 1:1 benzene:chloroform mixture was added, the organic layer was separated and added slowly to refluxing benzene (2x). The reaction was refluxed for 1 hr then concentrated. Chromatography using DCM and 20% MeOH in DCM gave 0.978 g (2.86 mmol) of (8S)-di-tert-butyl2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (B1) as a 4:1 (5S,8S:5S,8R) mixture of the diastereomers. 'H NMR (300 MHz, DMSO) 7.63 (m, 1 H), 4.29 - 4.09 (m, 1 H), 3.65 - 3.39 (m, 4H), 2.5 - 2.0 (m, 2H), 1.43 -1.18 (m, 18H).
[00330] Step C: (5S,8S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (Cl) p N
O
O
BocN
O
O

[00331] (8S)-di-tert-butyl2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (Bl) (0.189 g, 0.55 mmol, 1 eq.), Xantphos (4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene ) (17.4 mg, 0.03 mmol, 0.06 eq.), K3PO4 (148.6 mg, 0.7 mmol, 1.4 eq.) were combined in a sealable tube, evacuated, and purged with nitrogen 3 times. Dry 1,4-dioxane (20 mL) was -added- and-the-suspension was degassed -for 0.5- hrs. - Pd2(dba)3 (9.1 mg g, 0.01 mmol, 0.02 eq.) was added and the sealable tube was degassed for an additional 15 min.
Bromobenzene (53 pL, 0.5 mmol, 1 eq.) was added, the reaction was sealed and heated at 95 C
for 72 hrs (later optimization showed that 24 hrs were sufficient and additional equivalents of bromobenzene, up to 3 eq., gave higher yields). The reaction was cooled, filtered and flushed with DCM. Chromatography on the organic layer using hexanes and ethyl acetate with a conservative gradient separated the diastereomers to give 50 mg (0.12 mmol) of (5S,8S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (Cl). 'H NMR
(300 MHz, DMSO) 7.52 (d, 2 H), 7.43 - 7.37 (m, 2H), 7.14 (t, 1H), 4.22 - 4.07 (m, 3H), 3.85 (d, 1 H), 3.60 - 3.46 (m, 1 H), 2.8-2.65 (m, 1 H), 2.27 - 2.19 (m, 1 H), 1.42 (m, 18H).
[00332] Step D: (5S,8S)-tert-butyl2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8--carboxylate (D1) / ~

N I
i ~
O
HN O
O
Ok [00333] (5 S,8S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (C 1) (0.262 g, 0.626 mmol, I eq.) was added to a flask and cooled in an ice water bath. 1.0 M HCl in anhydrous ethyl acetate (6.26 mL, 6.26 mmo1,10 eq.) was added to the cooled intermediate, and the mixture was stirred at room temp. overnight.
The solvent was evaporated while the mixture was cold. Chromatography using DCM and 20%
MeOH in DCM gave 148 mg (466 mol) of (5S,8S)-tert-butyl 2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (D1). LC MS +: 319.17 at 1.76 min (10-90%, 3-5 min, Formic Acid).
[00334] Step E: (5S,8S)-tert-butyl7-((S)-2-(benzyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (El) p ~N
CbzHN >=O
N O
O
O Ok El [00335] L-Cbz-t-leu-OH (284 mg, 0.642 mmol, 1.4 eq.), EDC (123 mg, 0.642 mmol, 1.4 eq.) and HOBt (98 mg, 0.642 mmol, 1.4 eq.) were combined in DMF (0.9 mL) and stirred for 0.5 hrs at room temp. (5S,8S)-tert-butyl2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (D1) (146 mg, 0.459 mmol, 1 eq.) and DIEA (240 L, 1.377 mmol, 3 eq.) were added to the room temp. mixture, and the resulting mixture was stirred for 24 hrs at room temp. 1.0 M histamine (aq., 230 L, 0.5 eq.) was added to this mixture, and the resulting mixture stirred for 0.5 hrs. Ethyl acetate was added and the mixture was washed first with IN HCl then 1 M K2C03. Ethyl acetate was used for extraction, and this extraction process was repeated once. The combined organic layers were dried on Na2SO4 and concentrated.
Chromatography using hexanes and ethyl acetate gave (5S,8S)-tert-butyl 7-((S)-(benzyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (E1). LC MS +: 566.44 at 3.61 min (10-90%, 3-5 min, Formic Acid).
[00336] Step F: (5S,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (Fl) p N
HzN >= O
O
O
ON
~
O

Fl [00337] Palladium (10%) on carbon (100 mg) was wetted with MeOH (2.5 mL) and added to (5 S,8S)-tert-butyl 7-((S)-2-(benzyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (El) (323 mg, 411 mol, 1 eq.). The reaction was hydrogenated at 50 psi overnight at room temp., then filtered and concentrated to give (5S,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (F1) in quantitative yield. LC MS +:
432.39 at 1.94 min (10-90%, 3-5 min, Formic Acid).
j003381 Step G: (5S,8S)-tert-butyl 7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)=2-oxo-3-phenyl-l-oxa=3;7-diazaspiro [4.4] nonane-8-carboxylate (Gl) p N
HN >=O
N O
O

O ~<
O

[00339] Cyclohexylacetic acid (48 mg, 337 mol, 1.4 eq.), EDC (65 mg, 337 mol, 1.4 eq.) and HOBt (52 mg, 337 mol, 1.4 eq.) were combined in DMF (0.3 mL) and stirred for 0.5 .hrsyat room temp. (5S,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-1-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (Fl) (105 mg, 243 mol, 1 eq.) and DIEA
(118 L, 675 mol, 2.8 eq.) were added to this mixture and the resulting mixture was stirred for 24 hrs at room temp. 1.0 M histamine (aq., 120 L, 0.5 eq.) was added and the mixture was stirred for 0.5 h. Ethyl acetate was added and the mixture was washed with 1 N HCl then 1M K2CO3 and extracted with ethyl acetate (2x). The combined organic layers were concentrated and chromatography using hexanes and ethyl acetate gave 50 mg of (5S,8S)-tert-butyl 7-(( S )-2 -(2-cyclohexylacetamido)-3 , 3 -dimethylbutanoyl)-2 -oxo-3 -phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (G1). 'H NMR (300 MHz, DMSO) 7.90 (d, 1 H), 7.53 (d, 2 H), 7.44 - 7.37 (m, 2H), 7.14 (m, 1H), 4.47 (d, 1H), 4.30 - 3.99 (m, 3H), 3.89 - 3.83 (m, 1H), 3.18 (d, 1H), 2.79 - 2.67 (m, 1H), 2.27 - 2.00 (m, 3H), 1.7 - 1.55 (m, 5H), 1.45 -1.39 (m, 11 H), 1.2 - 1.1 (m, 4H), 0.98 - 0.91 (m, 9H). Later optimization showed that steps E
to G could be replaced by doing a single coupling on El with (S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoic acid. NMR confirmed that epimerization was not occurring.
[00340] Step H: (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (H1) p O
-4 ~- N
HN ~O
N O
O
O
OH

1003411 (5S,8S)-tert-butyl7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3--phenyl-1-oxa=3,7-diazaspiro[4.4]nonane-8-carboxylate-(G1-)-(70 rng, 126 mol, 1 eq.) was diluted in 1:1 DCM:TFA (2 mL) and stirred overnight at room temp. then concentrated to give (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-1-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (H1) in quantitative yield. LC
MS
500.41/498.55 at 3.09 min (10-90%, 3-5 min, Formic Acid).
[00342] Step I: (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (11) N
HN r>=O
N O

H

N
HO H

[00343] (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (20 mg, 87 mol, 1.4 eq.), EDC
(17 mg, 87 mol, 1.4 eq.) and HOBt (13 mg, 87 mol, 1.4 eq.) were combined in DMF (0.3 mL) and stirred at room temp. for 0.5 hrs. (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (H 1) (31 mg 62 mol, 1 eq.) and DIEA (33 L, 186 mol, 3 eq.) were added to the mixture and stirred at room temp. overnight. 1.0 M histamine (aq., 31 L, 0.5 eq.) was added and the resulting mixture was stirred for 0.5 hrs. Ethyl acetate was added and the mixture was washed first with 1 N HCl then with 1 M K2CO3; and then extracted with ethyl acetate. The combined organic layers concentrated and chromatography using DCM and 20% MeOH
in DCM gave 20 mg (30 mol) of (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (I1). LC MS 668.56/666.64 at 3.24 min (10-90%, 3-5 min, Formic Acid).
[00344] Step J: (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (Cmpd. No. 3) o N
HN = >= O
N O
O
O O
N ^
H N/~
O H
Cmpd No. 3 [00345] (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3S)-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (I1) (20 mg, 30 mol, 1 eq.) was diluted in DCM.
Dess Martin reagent (38 mg, 90 mol, 3 eq.) was added and the mixture was stirred for 3 hrs at room temp. Na2S2O3 (aq., 5 eq.) was added and the mixture was stirred for another 0.5 hrs.
The product was extracted with DCM. Chromatography on the combined organic layers using hexanes and ethyl acetate gave 15.9 mg (24 mol) (5S,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (compound no. 3). 1 H
NMR (300 MHz, CDC13) 7.5 (d, 2H), 7.4 - 7.32 (m, 2H), 7.2 - 7.12 (m, 2H), 6.9 (m, 1H), 6.17 (d, 1 H), 5.4 - 5.3 (m, 1 H), 4.77 (t, 1 H), 4.65 (d, 1 H), 4.42 (d, 1 H), 4.15 - 3.98 (m, 2H), 3.76 (d, 1 H), 2.83 - 2.75 (m, 1 H), 2.6 (m, 2H), 2.2 - 0.8 (m, 31 H), 0.6 (m, 2H).
Example 2: (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (Cmpd No. 5) [00346] Step A: (5R,8S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A2) o N-~

O N

[00347] Following the same procedure as Example 1 step C using (8S)-di-tert-butyl2-oxo-1-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (B1) (1.044 g, 3.05 mmol) gave 475 mg of (5R,8 S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A2). 'H NMR (300 MHz, DMSO) 7.52 (d, 2H), 7.43 - 7.37 (m, 2H), 7.14 (t, 1H), 4.37 - 4.3 (m, 1 H), 4.18 - 4.07 (m, 2H), 3.77 - 3.6 (m, 2H), 2.6 - 2.55 (m, 1 H), 2.45 -2.35 (m, 1 H), 1.42 (m, 18H).
[00348] Step B: (5R,8S)-tert-butyl2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B2) Q
N-~O
,0 HN
O

x [00349] Following the same procedure as Example 1 step D using (5R,8S)-di-tert-butyl-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A2) (145 mg, 0.346 mmol) gave 92 mg (0.289 mmol) of (5R,8S)-tert-butyl 2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B2). 'H NMR (300 MHz, DMSO) 7.54 (d, 2H), 7.41 -7.36 (m, 2H), 7.15 - 7.10 (m, 1 H), 4.08 (dd, 2H), 3.75 - 3.70 (m, 1 H), 2.90 (m, 1 H), 2.31 -2.26 (m, 1H), 1.44 - 1.39 (m, 9H).
[00350] Step C: (5R,8S)-tert-butyl 7-((,5)-2-(benzyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxylate (C2) ~O
N
O N
o CfO~-H N
O Q

[00351] Following the same procedure as Example 1 step E using (5R,8S)-tert-butyl 2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B2) (130 mg, 411 mol) gave (5R, 8 S)-tert-butyl 7-((S)-2-(benzyloxycarbonylamino)-3,3 -dimethylbutanoyl)-2-oxo-3 -phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C2). LC MS +: 566.44 at 3.61 min (10-90%, 3-5 min, Formic Acid).
[00352] Step D: (5R,8S)-tert-butyl 7-((S)-2=amino-3,3-dimethylbutanoyl)-2-oxo-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (D2) ~o N

'10 N
O

x [00353] Following the-same procedure as Example I step F, (5R,8S)-tert-butyl 7-((S)-2-(benzyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C2) gave (5R,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (D2). LC
MS +: 432.39 at 1.94 min (10-90%, 3-5 min, Formic Acid).

[00354] Step E: (5R,8S)-tert-butyl 7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxylate (E2) - ~o N

N
O

O H .

[00355] Following the same procedure as Example 1 step G using (5R,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (94 mg, 218 mol) gave 70 mg (126 mol) of (5R,8S)-tert-butyl 7-((S)-2-(2-cyclohexyl acetamido)-3, 3-dimethylbutanoyl)-2 -o xo-3 -phenyl-l-oxa-3 , 7-diazaspiro[4.4]nonane-8-carboxylate (E2). 'H NMR (300 MHz, DMSO) 7.85 (d, 1 H), 7.55 (d, 2 H), 7.44 - 7.37 (m, 2H), 7.13 (m, 1 H), 4.52 (m, 1 H), 4.38 (d, 1 H), 4.2 - 3.99 (m, 2H), 3.18 (d, 1 H), 2.6 (m, 1 H), 2.4 (m, 1 H), 2.2 - 1.94 (m, 3H), 1.7 - 0.8 (m, 29H).
[00356] Step F: (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (F2) QN
--~

N
O
HN O HO
O

[00357] Following the same procedure as Example I step H using using (5R,8S)-tert-butyl 7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (70 mg, 126 mol) gave (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (F2).
LC MS +/-: 500.41/498.55 at 3.09 min (10-90%, 3-5 min, Formic Acid).
[00358] Step G: (5R,8,S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (G2) QN
-~

N
O
O H O HN

~>-NH
O OH

[00359] Following the same procedure as Example 1 step 9 using (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7--diazaspiro[4.4]nonane-8-carboxylic acid (49 mg, 98 mol) and (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (31 mg, 137 mol, 1.4 eq.) gave 20 mg (30 mol) of (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (G2). LC
MS +/- : 668.56/666.64 at 3.24 min (10-90%, 3-5 min, Formic Acid).
[00360] Step H: (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3;7-diazaspiro[4.4]nonane-8-carboxamide (Cmpd No. 5) QN
-~

N

~>-NH

0 Cmpd No. 5 [00361] Following the same procedure as Example 1 step 10 using (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (20 mg, 30 mol) gave 14.4 mg (22 mol) of (5R,8S)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro [4.4]nonane-8-carboxamide (compound no. 5). 'H NMR (300 MHz, CDC13) 7.6 - 7.37 (m, 4H), 7.2 - 7.12 (m, 2H), 6.95 (m, 1 H), 6.17 (d, 1 H), 5.45 - 5.3 5 (m, 1 H), 4.75 (m, 1 H), 4.45 (d, 1 H), 4.3 5 (d, IH), 4.25 (d, 1 H), 4.15 - 3.98 (m, 2H), 3.05 - 2.97 (m, 1 H), 2.85 - 2.73 (m, 1 H), 2.3 (m, 1 H), 2.15 - 0.8 (m, 3 1 H), 0.6 (m, 2H).
Example 3: Cyclopentyl (S)-1-((5S,8S)-3-(3-chlorophenyl)-8-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro f 4.41 nonan-7-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Cmpd No. 7) [00362] Step A: (5S,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-1-oxa-3,7-diazaspiro [4.4] nonane-7,8-dicarboxylate (A3) ci O

N

O

[00363] Following the same procedure as Example 1 step 3 using (8S)-di-tert-butyl2-oxo-1-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (8S)-di-tert-butyl2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (B1) (224 mg, 0.654 mmol) and 3-chloro bromobenzene (230 L, 1.962 mmol, 3 eq.) gave 107 mg (0.236 mmol) of (5S,8S)-di-tert-butyl3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A3). 'H
NMR (300 MHz, DMSO) 7.68 (m, 1H), 7.55 - 7.38 (m, 2H), 7.2 (m, 1H), 4.23-4.05 (m, 3H), 3.8 - 3.7 (m, 1 H), 3.5 (m, 1 H), 2.7 - 2.6 (m, 1 H), 2.25 - 2.1 (m, 1 H), 1.4 (m, 18H).
[00364] Step B: (5S,8S)-methyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B3) CI

//O
N-(~
\O
HN
O
-~O

[00365] Saturated HCl in MeOH (5 mL) was added to (5S,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (107 mg, 0.236 mmol) and stirred at room temp. overnight. The solution was concentrated and chromatography using DCM and 20% MeOH in DCM gave 60 mg (0.193 mmol) of (5S,8S)-methyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B3). 'H
NMR (300 MHz, DMSO) 7.68 (s, 1 H), 7.6 - 7.4 (m, 2H), 7.23 (m, 1H), 4.62 (m, 1 H), 4.22 (m, 2H), 3.85 - 3.68 (m, 4H), 3.6 - 3.5 (m, 1 H), 2.9 - 2.7 (m, 2H). LC MS +:
311.14 at 1.65 min (10-90%, 3-5 min, Formic Acid).
[00366] Step C: (5S,8S)-methyl-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxyc arbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C3).

CI

N-~

N

HN O-O
O
O

1003671 Following the same procedure as Example I step G using (5S,8S)-methyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (30 mg, 97 mol) and (S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoic acid (28 mg, 116 mol, 1.2 eq.) gave 22 mg (41 mol) of (5S,8S)-methyl-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C3). 'H NMR (300 MHz, CDC13) 7.35 - 6.8 (m, 4H), 5.21 - 5.06 (m, 1 H), 4.86 (m, 1 H), 4.55 - 3.56 (m, 8H), 2.65 - 2.48 (m, 1 H), 2.0 (m, 1 H), 1.7 -"1.1 (m, 9H), 0.9 - 0.7 (m, 9H).
[00368] Step D: (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxylic acid (D3).
cl o O

N

H~ 0 HO
O

[00369] (5S,8S)-methyl-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C3) (22 mg, 41 mol, 1 eq.) was diluted with dry THF (0.5 mL) then 1 M LiOH (82 L, 82 mo1, 2 eq.) and MeOH (20 L) was added. The reaction was stirred overnight at room temp. and concentrated to give 10 mg (19 mol) of (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D3). LC MS +: 522.35 at 3.14 min (10-90%, 3-5 min, Formic Acid).
[00370] Step E: Cyclopentyl2-((5S,8S)-3-(3-chlorophenyl)-8-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonan-7-yl)-2-oxoethylcarbamate (D4).
ci o O

N
~ O
O~N" O HN
~NH
O OH

[00371] Following the same procedure as Example 1 step I using (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3 -dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D3) (10 mg, 20 mol) and (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (7 mg, 28 pmol) gave 5 mg (7.2 mol) of Cyclopentyl-(2S)-1-((5 S,8 S)-3-(3-chlorophenyl)-8-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonan-7-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (E3). LC MS +: 690.38 at 3.31 min (10-90%, 3-5 min, Formic Acid).
[00372] Step F: Cyclopentyl2-((5S,8S)-3-(3-chlorophenyl)-8-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonan-7-yl)-2-oxoethylcarbamate (Compound No. 7) CI
O
N-~

N
O

~NH

Cmpd No. 7 [00373] Following the same procedure as Example 1 step 10 using cyclopentyl 2-((5S,8S)-3-(3-chlorophenyl)-8-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonan-7-yl)-2-oxoethylcarbamate (D4) (5 mg, 7.2 mol) gave 0.9 mg (1.3 mol) of cyclopentyl-2-((5S,8S)-3-(3-chlorophenyl)-8-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4] nonan-7-yl)-2-oxoethylcarbamate (compound no. 7). LC MS +/-: 688.4/686.6 at 3.6 min (10-90%, 3-5 min, Formic Acid).
Example 4: (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-~diazaspirof4.41nonane-8-carboxamide_(Cmpd No. 9) [00374] Step A: (5R,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A4) ci ~o N
O
N
O

X

[00375] Following the procedure from Example 1 step C using (8S)-di-tert-butyl 2-oxo-1-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (2) (189 mg, 0.55 mmol, 1 eq.) and 3-chloro bromobenzene (60 l, 0.55 mmol, 1 eq.) gave 54 mg (119) of (5R,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A4). 'H
NMR

(300 MHz, DMSO) 7.68 (m, 1H), 7.55 - 7.38 (m, 2H), 7.2 (m, 1H), 4.38 - 4.28 (m, 1H), 4.2 -4.05 (m, 2H), 3.75 - 3.6 (m, 2H), 2.7 - 2.4 (m, 2H), 1.4 (m, 18H).
[00376] Step B: (5R,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B4) ci ~o N

HN

O

[00377] Following the procedure from Example 1 step D using (5R,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A4) (54 mg, 120 mol) gave 20 mg (57 mol) of (5R,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B4).
[00378] Step' C: - (5R;8S)-tert-buty1-34-(-3=chlorophenyl)-7=((S)-2=(2=cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxylate (C4) ci ~o N
O
N
O

O H x C4 [00379] Following the procedure from Example 1 step G using (5R,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B4) (20 mg, 54 mol, 1 eq.) and (S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoic acid (16 mg, 63 mol, 1.2 eq.) gave 16 mg (27 mol) of (5R,8S)-tert-butyl-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C4). LC MS +/-: 590.31/589.47 at 3.95 min (10-90%, 3-5 min, Formic Acid).
[00380] Step D: (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D4) ci o N
O

O

[00381] Following the procedure from Example 1 step H using (5R,8S)-tert-butyl-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C4) (16 mg, 27 mol) gave (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D4). LC MS +/-: 534.32/532.53 at 3.35 min (10-90%, 3-5 min, Formic Acid).
[00382] Step E: (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-1-oxa-3,7-diazaspiro [4.4] nonane-8-carboxamide (E4) ci o N-~
,0 N
O
N O HN
O H
~NH
O E

[00383] Following the procedure from Example 1 step I using (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-2-oxo-l-oxa-3, 7-diazaspiro[4.4]nonane-8-carboxylic acid (D4) (12 mg, 22 mol) and (3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide (7.3 mg, 33 mol) gave 10 mg (14 mol) of (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (E4). LC MS +/-: 702.54/700.74 at 3.68 min (10-90%, 3-5 min, Formic Acid).
[00384] Step F: (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (F4) ci o N

~>-Nq 0 Cmpd No. 9 [00385] Following the procedure from Example 1 step J using (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (E4) (10 mg, 14 mol) gave 2 mg (2.9 mol) of (5R,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (compound no. 9). IH
NMR
(300 MHz, CDC13) 7.62 (m, 1 H), 7.55 - 7.35 (m, 2H), 7.14 (m, 1 H), 6.9 (m, 1 H), 5.98 (m, 1 H), 5.4 (m, 1 H), 4.75 (m, 1 H), 4.5 - 4.25 (m, 3 H), 4.0 - 3.8 (m, 2H), 3.05 (m, 1 H), 2.8 (m, IH), 2.3 (m, 1 H), 2.15 - 1.9 (m, 2H) 1.7-0.8 (m, 30H), 0.6 (m, 2H).
Example 5: (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (Cmpd. No. 8) [003861 Step A: (5S,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro [4.41 nonane-7,8-dicarboxylate (A5).

CI
O

N-~
O
N

X O O
x [00387] Following the procedure from Example 1 step C using (8S)-di-tert-butyl2-oxo-1-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (B2) (189 mg, 0.55 mmol, 1 eq.) and 3-chloro bromobenzene (60 l, 0.55 mmol, 1 eq.) gave 48 mg (0.106 mmol) of (5S,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A5). 'H
NMR (300 MHz, DMSO) 7.68 (m, 1H), 7.55 - 7.38 (m, 2H), 7.2 (m, 1H), 4.23 -4.05 (m, 3H), 3.8 - 3.7 (m, 1 H), 3.5 (m, 1 H), 2.7 - 2.6 (m, 1 H), 2.25 - 2.1 (m, 1 H), 1.4 (m, 18H).
[00388] Step B: (5S,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (32).
cl /- \

o N-~
O
HN
O

x [00389] Following the procedure from Example 1 step 4 on (5S,8S)-di-tert-butyl 3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-7,8-dicarboxylate (A5) (48 mg, 106 mol).gave 34 mg (96 mol) of (5S,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B5).
[00390] Step C: (5S,8S)-tert-butyl-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxylate (33).

CI

O
~-N
0,,, N

/

O

[00391] Following the procedure from Example I step G using (5S,8S)-tert-butyl-3-(3-chlorophenyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (B5) (32 mg, 91 mol, 1 eq.) and (S')-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoic acid (25 mg, 100 mol, 1.2 eq.) gave 25 mg (42 mol) of (5S,8S)-tert-butyl-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C5). LC MS +/-: 590.31/589.47 at 3.95 min (10-90%, 3-5 min, Formic Acid).
[00392] Step D: (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D5).
ci o N

[00393] Following the procedure from Example 1 step H using (5S,8S)-tert-butyl-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C5) (25 mg, 42 mol) gave (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-2-oxo-l-oxa-3, 7-diazaspiro[4.4]nonane-8-carboxylic acid (D5). LC MS +/-: 534.32/532.53 at 3.39 min (10-90%, 3-5 min, Formic Acid).
[00394] Step E: (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-1-oxa-3,7-diazaspiro [4.4] nonane-8-carboxamide (E5) ci o ~-N
O1''', N
O
N H O H

HN-<

(HO~ O

[00395] Following the procedure from Example 1 step I using (5S,8S)-3-(3-chlorophenyl)-7-(( S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D5) (23 mg, 43 mol) and (3S)-3-amino-lV-cyclopropyl-2-hydroxyhexanamide (15 mg, 65 mol) gave 10 mg (14 mol) of (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (E5). LC MS +/-: 702.54/700.62 at 3.63 min (10-90%, 3-5 min, Formic Acid).
[00396] Step F: (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro [4.4] nonane-8-carboxamide (Cmpd. No. 8) ci o O
0-~ N N

H

Cmpd No. 8 [00397] Following the procedure from Example 1 step J using (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((3 S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (E5) (10 mg, 14 mol) gave 1.3 mg (1.9 mol) of (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-N-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxamide (compound no. 8). 'H
NMR
(300 MHz, CDC13) 7.6-7.28 (m, 3H), 7.13 (m, 2H), 6.9 (m, 1H), 6.03 (m, 1H), 5.33 (m, 1H), 4.8 - 4.75 (m, 1 H), 4.61 (m, 1 H), 4.46 - 4.4 (m, 1 H), 4.25 - 3.7 (m, 3H), 2.8 (m, 1 H), 2.68 -2.52 (m, 1 H), 2.2 - 1.9 (m, 2H), 1.9 - 0.8 (m, 30H), 0.6 (m, 2H).
Example 6: Cyclopentyl-(S)-1-((3S,5R)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-vlcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro f 4.41 nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Cmpd No. 1) and cyclopentyl (S)-1-((3S,5S)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro [4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Cmpd No. 4) [00398] Step A: (S)-di-tert-butyl-4-(2-ethoxy-2-oxoethylidene)pyrrolidine-1,2-dicarboxylate (A6) CO2Et BocN

O
O

[00399] To a solution of ((S)-di-tert-butyl 4-oxopyrrolidine-1,2-dicarboxylate (A1), 2.43 g, 8.5 mmol) in Toluene (20m1) was added (carboethoxy-methylene)triphenylphosporane (4.52 g, 13 mmol, 1.5 eq). The reaction was refluxed overnight, and concentrated to an oil. The oil was purified on silica (120g, hexane/ethyl acetate gradient) to afford (S)-di-tert-butyl-4-(2-ethoxy-2-oxoethylidene)pyrrolidine-1,2-dicarboxylate (A6) (1.00 g, 2.8 mmol) as a colorless oil. 'H NMR (DMSO) b1.20 (t, 3H), 1.40 (m, 18H), 2.54 (m, 2H), 3.30 (m, 2H), 4.10 (m, 3H), 4.71 (m, 0.5H), 5.62 (m, 0.5H).
[00400] Step B: (2S)-di-tert-butyl-4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)pyrrolidine-1,2-dicarboxylate (B6) EtO2C NO2 BocN

O

1004011 A solution of (S)-di-tert-butyl-4-(2-ethoxy-2-oxoethylidene)pyrrolidine-1,2-dicarboxylate (A6) (1.00 g, 2.8 mmol) in nitromethane (5ml) was treated with tetramethylguanidine (TMG, 0.35 g, 3.0 mmol, 1.1 eq). The reaction was refluxed for 3 hrs and concentrated to a yellow oil. The oil was purified on silica (hexane/ether gradient) to give (2S)-di-tert-butyl-4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)pyrrolidine-1,2-dicarboxylate (B6) (0.46 g, 1.1 mmol) as a mixture of diastereomers. 'H NMR (DMSO) 51.15 (t, 3H), 1.35 (m, 18H), 1.95 (m, 1H), 2.40 (m, 1H), 2.54 (m, 8H), 2.70 (s, 1.5H), 3.30 (s, 8.6H), 3.5-3.6 (m, 1H), 4.0-4.2 (m, 3H), 4.7-4.85 (m, 2H).
[00402] Step C: (2S)-di-tert-butyl 4-(aminomethyl)-4-(2-ethoxy-2-oxoethyl)pyrrolidine-1,2-dicarboxylate (C6) Et02C NHZ
BocN ~
O
O

[00403] The nitromethane adduct (2S)-di-tert-butyl-4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)pyrrolidine-1,2-dicarboxylate (B6) (1.24 g, 3.0 mmol) was dissolved in ethyl acetate (20m1), and to this was added 10% Pd/C (75mg, cat). The reaction was stirred under hydrogen atmosphere 2 days. The solution was filtered through celite and concentrated to an oil. The crude was purified on silica (1% to 20% MeOH/CH2ClZ gradient) to give (2S)-di-tert-butyl 4-(aminomethyl)-4-(2-ethoxy-2-oxoethyl)pyrrolidine-l,2-dicarboxylate (C6) (566 mg, 1.5 mmol) as a white foam. 'H NMR (DMSO) S 1.20 (t, 1 H), 1.4 (m, 18H), 1.8-1.9 (m, 1 H), 2.1-2.6 (m, 11 H), 3.0-3.5 (m, 5H), 3.9-4.2 (m, 2H), 7.61 (m, 0.5H).
[00404] Step D: 2-((5S)-3-(aminomethyl)-1,5-bis(tert-butoxycarbonyl)pyrrolidin-yl)acetic acid (D6) BocN ~
O
O

[00405] Ester (2S)-di-tert-butyl 4-(aminomethyl)-4-(2-ethoxy-2-oxoethyl)pyrrolidine- 1,2-dicarboxylate (C6) (560 mg, 1.45 mmol) was dissolved in THF (12m1). To this was added 1 N lithium hydroxide in water (2.9 ml, 2.9 mmol, 2.0 eq). The mixture was stirred at room temperature overnight. The reaction was cooled to 0 C and quenched with 1N
hydrochloric acid in water. The mixture was blown to dryness with N2 gas and stripped from acetonitrile several times to give 2-((5S)-3-(aminomethyl)-1,5-bis(tert-butoxycarbonyl)pyrrolidin-3-yl)acetic acid (D6) (1.45 mmol) as a glassy solid. 'H NMR (DMSO) b1.35 (m, 18H), 2.00 (m, 2H), 2.3 5(m, 2H), 2.80 (m, 1 H), 3.65 (m, 1 H), 4.00 (m, 1 H); LCMS for C
i 7H30N206 358.21 [M], found ES+ 359.21 [M+H]+ and ES- 357.21 [M-H]- at 1.8 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00406] Step E: (3S)-di-tert-butyl8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (E6) N O
BocN

O
O

[00407] To a solution of amino acid 2-((5S)-3-(aminomethyl)-1,5-bis(tert-butoxycarbonyl)pyrrolidin-3-yl)acetic acid (D6) (1.45 mmol) in CH2C12 (32m1) was added 1-hydroxybenzotriazole (HOBT, 196 mg, 1.45 mmol, 1 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 418 mg, 2.2 mmol, 1.5 eq), and N,N-Diisopropylethylamine (DIEA, 756 1, 4.4 mmol, 3.0 eq). The reaction was aged at room temp. overnight. The solution was diluted with CHZC12 (100 ml) and washed with water (100 ml), 1 N sodium hydroxide in water (100 ml) and brine (100 ml). The organics were dried over sodium sulfate, filtered, and concentrated with a second extract, to give crude product. This was purified on silica (1 % to 20% MeOH/CH2C12 gradient) to give spirolactam (3S)-di-tert-butyl 8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (E6) (295 mg, 868 mol). 'H NMR (CDC13) 61.35 (m, 18H), 1.91 (m, 2H), 2.3 (m, 3H), 3.2 (m, 2H), 3.3-3.5 (m, 2H), 4.10 (m, 1 H), 6.21 (m, 1 H); LCMS for C i 7H28N205 340.20 [M], found ES+ 341.2 [M+H]+, 285.2 [M+H, minus t-butyl]+, 285.2 [M+H, minus t-butyl, minus BOC]+ at 2.6 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00408] Step F: (3S,5S)-di-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (F6) N O
BocN
O
O

[00409] Spirolactam (3 S)-di-tert-butyl 8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (E6) (34 mg, 100 mol) was placed in a tube with CsCO3 (56 mg, 172 mol, 1.7 eq), Pd2dba3 (3.0 mg, 3.2 mol, 3.2%) and xanthphos ligand (5.7 mg, 9.8 mol, 9.8%).
Dioxane (1.0 ml) was added via syringe, followed by addition of bromobenzene (21 mg, 134 mol, 1.3 eq).
The sealed reaction was heated to 100 C for 16 hrs with efficient magnetic stirring. The reaction was diluted with CH2C12 (10 ml), filtered through celite and concentrated. The crude was purified on silica (1% to 6% MeOH/CH2C12 gradient) to give phenylated lactam (3 S,5 S)-di-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (F6) (32 mg, 77 mol) as a white foam. I H NMR (CDC13) 51.45 (m, 18H), 2.10 (m, 1H), 2.45 (m, 1H), 2.6-2.8 (m, 2H), 3.55 (m, 1 H), 3.75 (m, 2H), 4.25 (m, 1 H), 7.15 (m, 1 H), 7.35 (m, 2H), 7.55 (m, 2H); LCMS for C23H32N205 416.23 [M], found ES+ 417.23 [M+H]+ at 3.5 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[004101 Step G: (3S,5R)-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (Gla) and (3S,5S)-tert-butyl8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (Glb) N-~ N
+
HN HN
O

G6a G6b [00411] The phenylated lactam (3S,5S)-di-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (F6) (29mg, 70 mol) was taken up in dry ethyl acetate (1.0 ml) and treated with 1 M hydrochloric acid in ethyl acetate (1.0 ml, 1000 mol, 14 eq). The reaction was aged at room temperature for 24 hrs. The reaction was purged by bubbling N2 gas through it, until dry. The product was extracted with ethyl acetate and saturated sodium bicarbonate in water. The organics were washed with brine, dried over sodium sulfate, filtered and concentrated with a second extract to give crude product. The crude was purified on silica (1 % to 20% MeOH/CH2C12 gradient) to give (3 S,5R)-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (G6a) (6.1 mg, 19 mol, 27%). 1 H
NMR (CDC13) 51.45 (s, 9H), 2.00 (m, 1H), 2.40 (m, 1H), 2.5-2.8 (m, 4H), 3.0-3.2 (m, 2H), 3.88 (m, 3H), 7:20 (m, 1H), 7.38 (m,-2H), 7.6 (rn, 2H). The-minor-diastereomer (3S,5S)-tert-butyl 8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (G6b) (0.5 mg, 1.6 mol, 2.2%) was also obtained from the column. 'H NMR (CDC13) b1.45 (s, 9H), 2.25 (m, 1H), 2.45 (m, IH), 2.6-2.9 (m, 2H), 3.3 7(m, 1 H), 3.5-3.8 (m, 2H), 3.91 (m, IH), 4.25 (m, 1 H), 7.15 (m, IH), 7.32 (m, 2H), 7.55 (m, 2H).
[00412] Step H: (3S,5R)-tert-butyl-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (H6a) and (3S,5S)-tert-butyl 2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (H6b) N-~ N
,,.
+

~N 0 )-N O
O H O O O H O O
H6a H6b [00413] The amine (3S,5R)-tert-butyl-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-carboxylate (6.1 mg, 19 mol) in DMF (200 l) was treated with (5S,8S)-methyl-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3, 3 -dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (C3) (40 mol, 2 eq), 1-hydroxybenzotriazole (HOBT, 25 gmol, 1 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 40 mol, 2 eq), and N,N-Diisopropylethylamine (DIEA, 80 mol, 4 eq). The reaction was aged at room temp. overnight, quenched with I M histamine in water (40 l, 2 eq), and blown to dryness with nitrogen gas. The residue was extracted with ethyl acetate (2x5 ml) and 0.5N
hydrochloric.acid in water (2X2 ml), dried over sodium sulfate, filtered and concentrated to give (3 S,5R)-tert-butyl-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (H6a) (9.2 mg, 17 mol).
'H NMR
(CDC13) 51.08 (s, 9H), 1.4-1.9 (m, 20H), 2.09 (m, 1H), 2.40 (m, IH), 2.70 (s, 2H), 3.61 (m, 2H), 3.91 (m, IH), 4.20 (m, 2H), 4.45 (m, 1 H), 5.00 (m, 1 H), 5.20 (m, 1 H), 7.15 (m, 1 H), 7.35 (m, 2H), 7.60 (m, 2H); LCMS for C30H43N306 541.32 [M], found ES+ 564.3 [M+Na]+, 542.32 [M+H]+ at 3.7 minutes retention time, using 10-90% acetonitrile/water (with 0.1 %
formic acid buffer) gradient over 5 minutes. The minor diastereomer (3S,5S)-tert-butyl 2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (H6b) (5 mol) was obtained in similar fashion. LCMS
for C30H43N306 541.32 [M], found 542.32 [M+H]+ at 3.7 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00414] Step I: (3S,5R)-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (16a) and (3S,5S)-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (16b) N
,,.
+

, ~ - N OH /-N OH

16a 16b [00415] The protected proline (3S,5R)-tert-butyl-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylate (H6a) (9.2 mg, 17 mol) was dissolved in CHZC12 (1.0 ml) and treated with trifluoroacetic acid (0.3 ml) for 4 hrs at room temperature. The reaction was concentrated to dryness under high vacuum to give (3 S,5R)-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (16a) (17 mol). The minor diastereomer (3 S,5 S)-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3 -dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (16b) (5 mol) was obtained in similar fashion.
[00416] Step J: Cyclopentyl-(S)-1-((3S,5R)-3-((S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro [4.4] nonan-2-yl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (J6a) and cyclopentyl (S)-1-((3S,5S)-3-((S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (J6b) N N
0 N H OH + 0 N H OH
O-)-N N 0-1-N N
H 0 0 NH H 0 0 I~NH
\>
a 0 \> 6 O

J6a J6b [00417] The carboxylic acid (3S,5R)-2-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (16a) (17 mol) and (2R,3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide hydrochloride salt (warhead (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D3), 7.6 mg, 34 mol, 2 eq) were dissolved in DMF (1.0 ml). To this was added 1-hydroxybenzotriazole (HOBT, 4.6 mg, 34 mol, 2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 6.5 mg, 34 mol, 2 eq), and N,N-Diisopropylethylamine (DIEA, 12 l, 68 mol, 4 eq). The reaction was aged at room temperature overnight, concentrated to dryness and extracted with CH2ClZ (2x5 ml) and water (2 ml). The organics were dried over sodium sulfate, filtered and concentrated to crude. The crude was purified on silica (1%MeOH/CH2C12 to 20% MeOH/CH2C12 gradient) to give cyclopentyl-(S)-1-((3S,5R)-((S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (J6a) (4.1 mg, 6.3 mol).
LCMS for C35H51N507 653.38 [M], found ES+ 654.5 [M+H]+ and ES- 652.6 [M-H]", at 3.0 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes. The minor diastereomer cyclopentyl (S)-1-((3S,5S)-3-((S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (J6b) (3 mol) was obtained in similar fashion. LCMS for C35H51N507 653.38 [M], found ES+ 654.5 [M+H]+
and ES- 652.6 [M-H]", at 3.0 minutes retention time, using 10-90%
acetonitrile/water (with 0. 1%-formic acid buffer) gradient over 5 minutes.
1004181 Step K: Cyclopentyl-(S)-1-((3S,5R)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro [4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Cmpd No. 1) and cyclopentyl (S)-1-((3S,5S)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (Cmpd No. 4) Qo O
N-~ N
~ N N O N N O

O H O O NH O H O O NH
\>
6 \> 6 ro Cmpd No. 1 Cmpd No. 4 [00419] The hydroxy amide cyclopentyl-(S)-1-((3S,5R)-3-((S)-1-(cyclopropylamino)-2-hydroxy-l-oxohex an-3 -ylcarb amo yl)- 8-oxo-7-phenyl-2, 7-di azaspiro [ 4.4]
nonan-2 -yl )-3 , 3-dimethyl-l-oxobutan-2-ylcarbamate (J6a) (4.1 mg, 6.3 mol) was dissolved in CH2C12 (1.0 ml) and treated with Dess-Martin periodinane (DMP, 8.0 mg, 18.9 mol, 3 eq) for 3 hrs at room temp. The reaction was quenched with 1M sodium thiosulfate in water (32 l, 5 eq).
The reaction was loaded onto silica (eluted with ethyl acetate/hexane gradient) to give cyclopentyl-(S)-1-((3S,5R)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro [4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (compound no. 1) (3.1 mg, 4.8 mol) as a white solid. 'H NMR (CDC13) SS05m, 46H), 1.96 (m, 1 H), 2.25 (m, 1 H), 2.61 (m, 2H), 2.80 (s, 2H), 3.60 (dd, 2H), 4.00 (d, 1 H), 4.22 (m, 2H), 4.70 (m, 1 H), 4.90 (m, 1 H), 5.20 (d, 1 H), 5.3 5(m, 1 H), 6.90 (s, 1 H), 7.19 (m, 1H), 7.3 8(m, 2H), 7.65 (d, 1H). LCMS for C35H49N507 651.36 [M],.found ES+ 652.5 [M+H]+ and ES-650.6 [M-H]-, at 3.2 minutes retention time, using 10-90% acetonitrile/water (with 0.1%
formic acid buffer) gradient over 5 minutes. The minor diastereomer cyclopentyl (S)-1-((3 S,5 S)-3-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-ylcarbamoyl)-8-oxo-7-phenyl-2,7-diazaspiro[4.4]nonan-2-yl)-3,3-dimethyl-l-oxobutan-2-ylcarbamate (compound no.
4) (1.4 mg, 2 mol) was obtained in similar fashion. ~H NMR (CDC13) 50.52 (m, 2H), 7im, 14H), 1.0-2.0 (m, 22H), 2.10 (m, 1 H), 2.45-2.75 (m, 4H), 3.64 (t, 2H), 3.90 (d, 2H), 4.15 (d, 1 H), 4.60 (m, 1 H), 5.00 (m, 1 H), 5.13 (d, 1 H), 5.25 (m, 1 H), 6.80 (m, 1 H), 7.10 (m, 2H), 7.30 (m, 4H), 7.50 (m, 2H), 7.90 (m, 1 H); LCMS for C35H49N507 651.36 [M], found ES+
652.5 [M+H]+ arnd ES- 650:6 -[M-H]-, at 3.2 minutes retention time, using 10-90%
acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
Example 7: (3S,5R)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspirof4.4lnonane-3-carboxylate (Cmpd No. 2) and (3S,5S)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro(4.41 nonane-3-carboxylate (Cmpd No. 6) [00420] Step A: (3S,5S)-di-tert-butyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7a) and (3S,5R)-di-tert-butyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7b) ~
ci ~ / p p N-~ N
~N ~N
Boc p Boc p A7a A7b [00421] Spirolactam (5S,8S)-tert-butyl7-((S)-2-amino-3,3-dimethylbutanoyl)-2-oxo-3-phenyl-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylate (F1) (51 mg, 150 mol) was placed in a tube with CsCO3 (70 mg, 215 mol, 1.4 eq), Pd2dba3 (6.6 mg, 7.2 mol, 4.8%) and xanthphos ligand (13.5 mg, 23.3 mol, 15.5%). Dioxane (1.5 ml) was added via syringe, followed by addition of 1-bromo-3-chlorobenzene (46mg, 240 mol, 1.6 eq). The sealed reaction was heated to 100 C for 17 hrs with efficient magnetic stirring. The reaction was diluted with CH2C12 (10 ml), filtered through celite and concentrated. The crude was purified on silica (1 % to 6% MeOH/CH2C12 gradient) to give the product as a mixture of diastereomers. The diastereomers were separated on silica (10% to 50%
ether/hexane) to give (3S,5S)-di-tert-butyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7a) (45.7 mg, 101 mol). 'H NMR (CDC13) b1.48 (m, 18H), 2.09 (m, 1H), 2.45 (m, 1 H), 2.70 (q, 2H), 3.50 (m, 1 H), 3.70 (m, 3H), 4.28 (m, 1 H), 7.15 (d, 1 H), 7.30 (m, 1 H), 7.50 (d, 1 H), 7.65 (d, 1 H); LCMS for C23H31N205C1450.19 [M], found ES+
451.19 [M+H]+ at 3.7 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes. The minor diastereomer, (3S,5R)-di-tert-butyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7b) (15.2 mg, 34 mol) was also obtained. 'H NMR (CDC13) S 1.48 (m, 18H), 2.09 (m, 1 H), 2.45 (m, 1 H), 2.65 (s, 2H), 3.4-3.95 (m, 4H), 4.25 (m, 1H), 7.15 (d, 1 H), 7.1-7.7 (m, 4H); LCMS for 450.19 [M], found ES+ 451.19 [M+H]+ at 3.8 minutes retention time, using 10-90%
acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00422] Step B: (3S,5R) methyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7a) and (3S,5S)-methyl7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3-carboxylate (B7b).

CI Q 0 CI \ ~ O
N N
+
HN O> HN
O

B7a B7b [00423] The arylated lactam (3S,5S)-di-tert-butyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7a) (45.7 mg, 101 mol) was taken up in 1 M HCl in ethyl acetate (1.0 ml, 10 eq). The reaction was aged at room temperature overnight. The reaction was purged by bubbling N2 gas through it, until dry. The product was then taken up in a saturated solution of hydrochloric acid in methanol (2 ml), and aged at room temperature overnight. The reaction was blown dry with nitrogen gas and dried in-vacuo to give (3S,5R) methyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7a) (35.2 mg, 102 mol). LCMS for C15H17N203C1308.09 [M], found ES+ 309.2 [M+H]+ and 311.1 [M+H]+ (for the chlorine isotope) at 2.0 minutes retention time, using 5-45%
acetonitrile/water (with 0.1 % formic acid buffer) gradient over 7 minutes.
The minor diastereomer (3 S,5R)-di-tert-butyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-2,3-dicarboxylate (A7b) (15.2 mg, 34 mol) was treated similarly to give (3S,5S)-methyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7b) (12.4 mg, 36 mol).
LCMS for C15H17N2O3C1308.09 [M], found ES+ 309.2 [M+H]+ and 311.1 [M+H]+ (for the chlorine isotope) at 2.2 minutes retention time, using 5-45%
acetonitrile/water (with 0.1 %
formic acid buffer) gradient over 7 minutes.
[00424] Step C: (3S,5R)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3-carboxylate (C7a) and (3S,5S)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chloro phenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7b) ~ ~
CI ~ ~ O CI ~ ~ 0 N-~ N
+
O N O N ~
~NH O ~-NH O
O O ~0 O 0 C7a C7b [00425] Proline (3S,5R) methyl-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (B7a) (35.2 mg, 102 mol) in DMF (0.7 ml) was treated with (S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoic acid (33.3 mg, 144 mol, 1.2 eq), 1-hydroxybenzotriazole (HOBT, 16.5 mg, 122 mol, 1.2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 63.3 mg, 330 mol, 3.2 eq), and N,N-Diisopropylethylamine (DIEA, 86 l, 68 mol, 4 eq) for three days at room temp. The reaction was quenched with 1M histamine in water (381 l, 3.7 eq) and blown to dryness with N2 gas. The residue was extracted with ethyl acetate (2X 10 ml) and 0.5 N
hydrochloric acid (2X5 ml). The organics were washed with 1 M potassium carbonate in water (2X2ml), dried over sodium sulfate, filtered and concentrated. The product was purified on silica (ethyl acetate/hexane gradient) to give (3S,5R)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3 -dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3 -carboxylate (C7a) (36.9mg, 70.6 mol). 'H NMR (CDC13) 51.07 (s, 9H), 1.39 (s, 9H), 1.60 (s, 1H), 2.10 (m, 1 H), 2.45 (m, 1 H), 2.71 (s, 2H), 3.60 (m, 2H), 3.75 (s, 3H), 3.99 (d, 1 H), 4.13 (d, 1 H), 4.30 (d, 1 H), 4.60 (t, 1 H), 5.08 (d, 1 H), 7.15 (m, 1 H), 7.29 (m, 2H), 7.60 (m, 2H); LCMS for CZ6H36N306C1 521.23 [M], found ES+ 522.23 [M+H]+ and 422.23 [M+H-Boc]+ at 3.5 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes. The minor diastereomer (3S,5S)-methyl 7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[.4.4]nonane-3-carboxylate (B7b) (12.4 mg, 36 mol) was reacted similarly to give (3S,5S)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chloro phenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7b) (9.4 mg, 18 mol, 50%).
LCMS for C26H36N306C1 521.23 [M], found ES+ 522.23 [M+H]+ and 422.23 [M+H-Boc]+ at 3.5 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00426] Step D: (3S,5R)-methyl 2-((S)-2-amino-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (D7a) and (3S,5S)-methyl 2-((S)-2-amino-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3-carboxylate (D7b) ~
CI ~ ~ p CI ~ ~ p N-~ N
+

N N
HZN O O

D7a D7b [00427] The compound (3S,5R)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7a) (36.9mg, 70.6 mol) was dissolved in CH2C12 (1.0 ml) and treated with trifluoroacetic acid (0.3 ml) for 6 hrs at room temperature. The reaction was concentrated to dryness to give (3 S, 5 R)-methyl2-((S)-2-amino-3,3 -dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2, 7-diazaspiro[4.4]nonane-3-carboxylate (D7a) (70.6 mol). The minor diastereomer (3S,5S)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3 -dimethylbutanoyl)-7-(3-chloro phenyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3 -carboxyl ate (C7b) (9.4 mg, 18 mo1). was treated similarly to give (3S,5S)-methyl 2-((S)-2-amino-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3 -carboxylate (D7b) (18 mol, 100%).
j004281 Step E: (3S,5R)-methyl-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3;3-diniethy[ butanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane=3=ca"rboxylate (E7a) arid (3S,5S)-methyl7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (E7b).
~
CI ~ ~ p CI ~ ~ p N-~ N
,~.

O N O O N C/
N
N 0~
(D--KH p p H O p E7a E7b [00429] The amine (3S,5R)-methyl2-((S)-2-amino-3,3-dimethylbutanoyl)-7-(3-chlorophenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (D7a) (70.6 mol) was taken up in CH2Cl2 (2.0 ml) and treated with 2-cyclohexylacetic acid (20.0 mg, 141 mol, 2.0 eq), 1-hydroxybenzotriazole (HOBT, 9.5 mg, 70 mol, 1.0 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 27.1 mg, 141 mo1, 2.0 eq), and N,N-diisopropylethylamine (DIEA, 74 l, 424 pmol, 6 eq) at room temp.
overnight. The reaction was extracted with CH2C12 (2X10 ml) and water (5 ml). The organics were washed with brine, dried over sodium sulfate, filtered and concentrated to give crude. The product was purified on silica (ethyl acetate/hexane gradient) to give (3S,5R)-methyl-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethyl butanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (E7a) (12.8 mg, 23 mol). 'H NMR (CDC13) 50.85-1.4 (m, 17H), 1.67 (m, 8H), 2.10 (m, 4H), 2.45 (m, 1H), 2.70 (d, 2H), 3.65 dd, 2H), 3.79 (s, 3H), 3.92 (d, 1 H), 4.30 (d, 1 H), 4.50 (d, IH), 4.60 (t, 1 H), 6.00 (d, IH), 7.15 (d, 1 H), 7.30 (m, 2H), 7.50 (m, 1 H), 7.70 (s, 1 H); LCMS for C29H40N305C1 545.26 [M], found ES+
546.3 [M+H]+
and 548.4 [M+H]+ (for the chlorine isotope) and ES- 544.3 [M-H]- at 3.6 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes. The minor diastereomer (3S,5S)-methyl-2-((S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoyl)-7-(3-chloro phenyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (C7b) (18 mol) was treated similarly to give (3S,5S)-methyl 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro [4.4]nonane-3-carboxylate (E7b) (6.6 mg, 12 mol). LCMS for C29H40N305C1 545.26 [M], found ES+ 546.3 [M+H]+ at 3.6 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00430] Step F: (3S,5R)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (F7a) and (3S,5S)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (F7b).

ci \ / o ci \ / o N-~ N
~..
+

N OH N OH

F7a F7b [00431] The methyl ester (3S,5R)-methyl-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethyl butanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (E7a) (12.8 mg, 23 mol) was taken up in THF (1.0 ml) and MeOH (50 l) was added. To this was added 1N LiOH in water (46 l, 2.0 eq). The reaction was stirred at room temperature overnight and quenched at 0 C with 1N HCI in water (46 1, 2.0 eq).
The reaction was concentrated to dryness to give (3S,5R)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3 -carboxylic acid (F7a) (23 mol). LCMS for C28H38N305C1 531.25 [M], found ES+ 532.3 [M+H]+
and ES- 530.5 [M-H]" at 3.2 minutes retention time, using 10-90%
acetonitrile/water (with 0.1%
formic acid buffer) gradient over 5 minutes. The minor diastereomer (3S,5S)-methyl 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (E7b) (6.6 mg, 12 mol) was treated similarly to give (3 S,5 S)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (F7b) (12 mol). LCMS for 531.25 [M], found ES+ 532.25 [M+H]+ and ES- 530.25 [M-H]- at 3.3 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00432] Step G: (3S,5R)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (G7a) and (3S,5S)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3-carboxylate (G7b) o cI-Q O
N-~ +
.~
O N O OH N N O OH
A

NH
O
NH O
"">

G7a G7b [00433] The carboxylic acid (3S,5R)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (F7a) (23 mol) and (2R,3S)-3-amino-N-cyclopropyl-2-hydroxyhexanamide and hydrochloride salt (warhead (5S,8S)-3-(3-chlorophenyl)-7-((S)-2-(cyclopentyloxycarbonylamino)-3,3-dimethylbutanoyl)-2-oxo-l-oxa-3,7-diazaspiro[4.4]nonane-8-carboxylic acid (D3), 20.6 mg, 92 mol, 4 eq) were dissolved in CHZC12 (2.0 ml). To this was added 1-hydroxybenzotriazole (HOBT, 12.2 mg, 46 mol, 2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 17.6 mg, 92 mo1, 4 eq), and N,N-Diisopropylethylamine (DIEA, 50 l, 288 mol, 12.5 eq). The reaction was aged at room temp. overnight, and extracted with CHZC12 (2X10 ml) and water (2 ml). The organics were dried over sodium sulfate, filtered and concentrated to crude. The crude was purified on silica (1%MeOH/CH2C12 to 20% MeOH/CH2C12 gradient) to give (3S,5R)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro [4.4]
nonane-3 -carboxylate (G7a) (12.2 mg, 17.4 mol). LCMS for C37H54C1N506 699.38 [M], found ES+
700.5 [M+H]+ and ES" 698.7 [M-H]-, at 3.3 minutes retention time, using 10-90%
acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
The minor diastereomer (3S,5S)-7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylic acid (F7b) (12 mol) was treated similarly to give (3S,5S)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3 -chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3 -dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (G7b) (8.5 mg, 12 mol). LCMS for 699.38 [M], found ES+ 700.5 [M+H]+ and ES- 698.7 [M-H]-, at 3.3 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00434] Step H: (3S,5R)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (Cmpd No. 2) and (3S,5S)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro [4.4] nonane-3-carboxylate (Cmpd No. 6) CI
- -(~C N 0 N-\ +

p OJ- N
N H O O
H C C NH
NH
O
)o\

Cmpd No. 2 Cmpd No. 6 [00435] The hydroxy amide (3S,5R)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (G7a) (12.2 mg, 17.4 mol) was dissolved in CH2Cl2 (2.0 ml) and treated with Dess-Martin periodinane (DMP, 22.0 mg, 52 mol, 3 eq) for 3 hrs at room temperature. The reaction was quenched with 1 M sodium thiosulfate in water (87 l, eq). The reaction was loaded onto silica and eluted with an ethyl acetate/hexane gradient, to give (3S,5R)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (Cmpd No. 2) (12.1 mg, 17.3 mol) as a white solid. 'H NMR (CDC13) S529m, 44H), 3.51 (d, 2H), 3.91 (d, 1 H), 4.21 (d, 1 H), 4.44 (d, 1 H), 4.59 (m, 1 H), 5.22 (m, 1 H), 5.92 (d, 1 H), 6.81 (s, 1 H), 7.09 (m, 1 H), 7.20 (m, 4H), 7.50 (d, 1 H), 7.60 (s, 1 H); LCMS for C37H52C1N506 697.36 [M], found ES+ 698.5 [M+H]+ and ES" 696.6 [M-H]", at 3.6 minutes retention time, using 10-90% acetonitrile/water (with 0.1 % formic acid buffer) gradient over minutes. The minor diastereomer (3S,5S)-((3S)-1-(cyclopropylamino)-2-hydroxy-l-oxohexan-3-yl) 7-(3-chlorophenyl)-2-((S)-2-(2-cyclohexylacetamido)-3,3-dimethylbutanoyl)-8-oxo-2,7-diazaspiro[4.4]nonane-3-carboxylate (G7b) (8.5 mg, 12 mol) was treated similarly to give (3S,5S)-((S)-1-(cyclopropylamino)-1,2-dioxohexan-3-yl) 7-(3-chlorophenyl)-2-((S )-2 -(2 -cyclohexyl acetamido)-3, 3-dimethylbutanoyl)- 8-oxo-2, 7-diazaspiro[4.4]nonane-3-carboxylate (Cmpd No. 6) (7.8 mg, 11 mol, 93%). 'H
NMR
(CDC13) S528m, 45H), 3.65 (m, 2H), 3.90 (m, 2H), 4.49 (m, 2H), 4.60 (m, 1H), 5.28 (m, 1H), 5.95 (m, 1H), 6.81 (s, 1H), 7.0-7.8 (m, 10H); LCMS for C37H52C1N506 697.36 [M], found ES+ 698.5 [M+H]+ and ES" 696.6 [M-H]-, at 3.6 minutes retention time, using 10-90%
acetonitrile/water (with 0.1 % formic acid buffer) gradient over 5 minutes.
[00436] Reagents and conditions: (1) 1-bromo-3-chlorobenzene, Pd2dba3/
xanthphos/CsCO3, dioxane, 100 C, 16 h, 90% (3:1 mixture); (m) HCI(g), EtOAc, 18 h, then HChg), MeOH, 24 h, 100%; (n) (S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoic acid, HOBT/EDC/DIEA, DMF, 3 days, 69%; (o) TFA, CH2C12, 6 h, 100%; (p) 2-cyclohexylacetic acid, HOBT/EDC/DIEA, CH2C12,18 h, 67%; (q) LiOH, THF, MeOH, 18 h, 100%; (r) (2R,3S)-amino-N-cyclopropyl-2-hydroxyhexanamide hydrochloride, HOBT/EDC/DIEA, CH2C12, h, 76%; (s) Dess-Martin Periodinane, CH2C12, 3 h, 99%.
VI. ASSAYS FOR DETECTING AND MEASURING INHIBITION
PROPERTIES OF COMPOUNDS
A. HCV Enzyme Assays 1. Construction and Expression of the HCV NS3 Serine Protease Domain [00437] A DNA fragment encoding residues Ala'-Ser"l of the HCV NS3 protease (GenBank CAB46913) was obtained by PCR from the HCV Conl replicon plasmid, 1377neo/NS3-3'/wt (re-named as pBR322-HCV-Neo in this study) [V. Lohmann et al., Science, 285, pp. 110-113 (1999)] and inserted into pBEVI l(S. Chamber, et al., personal communication) for expression of the HCV proteins with a C-terminal hexa-histidine tag in E.
coli. All constructs were confirmed by sequencing.

[00438] The expression constructs for the HCV NS3 serine protease domain was transformed into BL21/DE3 pLysS E. coli cells (Stratagene). Freshly transformed cells were grown at 37 C in a BHI medium (Difco Laboratories) supplemented with 100 g/ml carbenicillin and 35 g/ml chloramphenicol to an optical density of 0.75 at 600 nm. Induction with 1 mM IPTG
was performed for four hrs at 24 C. The cell paste was harvested by centrifugation and flash frozen at -80 C prior to protein purification. All purification steps were performed at 4 C.
Next, 100.g of cell paste was lysed in 1.5 L of buffer A(50 mM HEPES (pH 8.0), 300 mM
NaCI, 0.1% n-octyl-(3-D-glucopyranoside, 5 mM (3-mercaptoethanol, 10% (v/v) glycerol) and stirred for 30 minutes. The lysate was homogenized using a Microfluidizer (Microfluidics, Newton, Mass.), followed by ultra-centrifugation at 54,000 x g for 45 minutes.
Imidazole was added to the supernatant to a final concentration of 5 mM along with 2 mL
of Ni-NTA
resin pre-equilibrated with buffer A containing 5 mM imidazole. The mixture was rocked for three hrs and washed with 20 column volumes of buffer A plus 5 mM imidazole.
The HCV
NS3 protein was eluted in buffer A containing 300 mM imidazole. The eluate was concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, pre-equilibrated with buffer A. The appropriate fractions of the purified HCV protein were pooled and stored at -80 C.
2. HCV NS3 Protease Domain Peptide Cleava eg Assay [00439] This assay is a modification of that described by Landro, et al.
(Landro JA, Raybuck SA, Luong YC, O'Malley ET, Harbeson SL, Morgenstern KA, Rao G and Livingston DL.
Biochemistry 1997, 36, 9340-9348), and uses a peptide substrate (NS5AB), based on the NS5A/NS5B cleavage site for genotype la HCV. The substrate stock solution (25 mM) was prepared in DMSO containing 0.2 M DTT and stored at -20 C. A synthetic peptide cofactor (KK4A) was used as a substitute for the central core region of NS4A. Peptide sequences are shown in the table below. The reaction was performed in a 96-well microtiter plate format using 25 r1M to 50 r1M HCV NS3 protease domain in buffer containing 50 mM
HEPES pH
7.8, 100 mM NaC1, 20% glycerol, 5 mM DTT and 25 M KK4A. The final DMSO
concentration was no greater than 2% v/v. Reactions were quenched by addition of trifluoroacetic acid (TFA) to yield a final concentration of 2.5%.

Peptide Sequences Used with HCV NS3 Protease Domain PEPTIDE SEQUENCE
NS5AB NH2-EDVV-(alpha)Abu-CSMSY-COOH [SEQ ID NO:2]
KK4A NH2-KKGSVVIVGRIVLSGK-COOH [SEQ ID NO:3]

[00440] The SMSY product was separated from substrate and KK4A using a microbore separation method. The instrument used was a Agilent 1100 with a G 1322A
degasser, either a G 1312A binary pump or a G 1311 A quaternary pump, a G 1313A autosampler, a column thenmostated chamber and a G1315A diode array detector. The column was a Phenomenex Jupiter, 5 gm C 18, 300 A, 150x2 mm, P/O OOF-4053-B0, with a flow-rate of 0.2 mL/min. The column thermostat was at 40 C. Mobile phases were HPLC grade H20/0.1 %
TFA (solvent A) and HPLC grade CH3CN/0.1 % TFA (solvent B). The SMSY product peak was quantitated using the data collected at 210 r1M.
Construction and Expression of NS3=4A Protease 1004411 Using standard recombinant DNA techniques, a cDNA fragment encoding the sequence for NS3 and NS4A, residues Ala1027 to Cys1711 from the HCV sub-type strain la, containing an N-terminal hexa-histidine sequence, was cloned into the baculoviral transfer vector pVL1392 (Webb NR and Summers MD (1990) Expression of proteins using recombinant baculoviruses, Techniques 2:173-188). Recombinant baculovirus containing NS3=4A was'produced by co-transfection of pVL1392-His-NS3=4A with linearized Autographa califomica nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugoperda (Sf9) insect cells. The transfected insect cells containing recombinant baculovirus clones were subsequently isolated by plaque purification. High-titer clonal baculovirus was routinely used to infect SO insect cells for protein production. In production, Sf9 cells were grown at 27 C until they reached a density of 2.0-x106 cells/ml. At this point, the insect cells were infected with virus. After 72 hrs or when the cell viability was between 70-80%
the culture was harvested and the cells were ready for purification.
3. Purification of NS3=4A Protein [00442] The NS3=4A protein (SEQ ID NO:1) was purified as follows. Cell paste was thawed in at least five volumes of Lysis Buffer (50 mM NaZHPO4 pH 8.0, 10%
Glycerol, 300 mM NaCI, 5 mM (3-mercaptoethanol, 0.2 mM PMSF, 2.5 gg/ml Leupeptin, 1.0 g/ml E64, 2.0 g/ml Pepstatin) per gram of cell paste. The cell paste was then homogenized on ice using a Dounce homogenizer. The cells were next mechanically disrupted by passing once through a microfluidizer (Microfluidics Corporation, Newton, MA), and the cell lysate was collected on ice. The cell lysates was centrifuged at 100,000 x g for 30 minutes at 4 C and the supernatants were decanted. Optionally, the pellets were resuspended in wash buffer (Lysis Buffer + 0.1 %(3-octyl glucopyranoside), homogenized using a Dounce homogenizer and centrifuged at 100,000 x g for 30 minutes at 4 C. Insoluble NS3=4A was extracted from the pellets by resuspending in Extraction Buffer (Lysis Buffer + 0.5% lauryl maltoside) using 2.5 ml/g cell paste. The mixture was homogenized using a Dounce homogenizer and mixed at 4 C for three hrs or more. The mixture was centrifuged at 100,000 x g for 30 minutes at 4 C. The supernatants were decanted and pooled.
[00443] The NS3=4A protein was further purified using Nickel-NTA metal affinity chromatography. Imidazole from a 2 M stock, pH 8.0, solution was added to the pooled supernatants so that the final concentration of imidazole was 10 mM. The supernatants were incubated batchwise overnight at 4 C with Nickel-NTA affinity resin that had been pre-equilibrated with Lysis Buffer + 10 mM imidazole. 1 ml of resin per 5 g of expected NS3-4A was used. The resin was next settled by gravity or by centrifugation at 500 x g for five minutes. The resin was next poured into a gravity flow column and washed with 10 or more column volumes of Nickel Wash Buffer (Lysis Buffer + 0.1% lauryl maltoside +
10 mM
imidazole). The column was next eluted with three to four column volumes of Nickel Elution Buffer (Nickel Wash Buffer + 300 mM imidazole). The elution fractions were collected on ice and evaluated using SDS-PAGE. To prevent NS3-4A proteolysis, 100 gM DFP
protease inhibitor was added to gel samples before adding SDS sample buffer and boiling. The peak fractions were pooled and protein concentration was determined by measuring absorbance at 280 rlm and by dividing by the extinction coefficient (e), which for NS3=4A is 1.01.
[00444] The NS3=4A was purified further using gel filtration chromatography. A
Superdex 200 26/60 column was equilibrated with Superdex Buffer (20 mM HEPES pH 8.0, 10%
glycerol, 300 mM NaCI, 10 mM (3-mercaptoethanol, 0.05% lauryl maltoside) at a rate of 3 ml/min. The nickel purified NS3=4A was concentrated in a Centriprep 30 to greater than 2 mg/ml, if necessary, and was filtered through a 0.2 gm syringe filter and up to 10 ml was loaded onto the Superdex 200 column. After 0.3 column volumes passed through, 4-5 ml fractions were collected. Fractions were evaluated by SDS-PAGE. NS3=4A protein elutes in two peaks. Peak 1 contains aggregated NS3=4A and peak 2 contains active protein. The fractions of peak 2 were pooled, aliquoted and frozen at -70 C.
Analysis of NS3 94A protein.

ANALYSIS ENTIRE PROTEIN
Length 695 amino acids Molecular Weight 74,347.78 1 microgram 13.450 picot moles ANALYSIS ENTIRE PROTEIN
Molar Extinction Coefficient 73430 1 A280 corresponds to 1.01 mg/ml Isoelectric Point 6.50 Charge at pH 7 -3.58 4. NS3 Peptide Cleavage Assay [00445] This assay follows the cleavage of a peptide substrate by full-length hepatitis C viral protein NS3=4A. One of three peptide substrates based on the NS5A/NS5B
cleavage site for genotype 1 a HCV is used to measure enzyme activity. All substrate stock solutions (25 mM) were prepared in DMSO containing 0.2M DTT and stored at -20 C. A synthetic peptide cofactor (NS4A Peptide) was used to supplement NS4A. Peptide sequences are shown below. The hydrolysis reaction was perfon ned in a 96-well microtiter plate format using 100 r1M to 125 r1M HCV NS3=4A in buffer containing 50 mM HEPES pH 7.8, 100 mM
NaCl, 20% glycerol, 5 mM DTT and 25 M NS4A Peptide. The final DMSO concentration was no greater than 2% v/v. Reactions using NS5AB or NS5AB-EDANS as substrate were quenched by the addition of 10% trifluoroacetic acid (TFA) to yield a final TFA
concentration of 2.5%. Reactions using FITC-NS5AB-1 as substrate were quenched by the addition of 0.4M formic acid to yield a final concentration of 0.08M acid.
1004461 Enzymatic activity was assessed by separation of substrate and products by reverse phase HPLC. The instrument used was a Agilent 1100 with a G1322A degasser, either a G1312A binary pump or a G 1311 A quatemary pump, a G 1313 A autosampler, a column thermostated chamber, a G1321A fluorescence detector and a G1315A diode array detector. The column thermostat was at 40 C. For substrate NS5AB the column was a Phenomenex Jupiter, 5 m C 18, 300 A, 150x2 mm, P/O OOF-4053-B0, with a flow-rate of 0.2 mL/min using HPLC grade H20/0.1% TFA (solvent A) and HPLC grade CH3CN/0.1% TFA
(solvent B) as mobile phases. The C-terminal product peak (NH2-SMSY -COOH) was quantitated using the absorbance data collected at 210 rlm. For substrate the column was a Phenomenex Aqua, 5 m C 18, 125 A, 50x4.6 mm, P/O OOB-4299-E0, with a flow-rate of 1.0 mL/min using HPLC grade H20/0.1% TFA (solvent A) and HPLC
grade CH3CN/0.1 % TFA (solvent B) as mobile phases. The C-terminal product peak (NH2-SMSYT-Asp(EDANS)-KKK-COOH) was quantitated using the fluorescence data collected at 350 rlm excitation / 490 rlm emission. For substrate FITC-NS5AB-1 the column was a Phenomenex Prodigy, 5 m ODS(2), 125 A, 50x4.6 mm, P/O OOB-3300-E0, with a flow-rate of 1.0 mL/min using 10 mM sodium phosphate pH 7.0 in HPLC grade H20 (solvent A) and 65% HPLC Grade CH3CN / 35% 10 mM sodium phosphate pH 7.0 in HPLC grade H20 (solvent B) as mobile phases. The N-terminal product peak (FITC-Ahx-EDVV-(alpha)Abu-C-COOH) was quantitated using the fluorescence data collected at 440 nm excitation / 520 nm emission. Alternatively, the ratio of N-terminal product to unreacted FITC-substrate was determined using a Caliper LabChip 3000 with detection at 488 nm excitation /
530 nm emission, using a chip buffer of 100 mM Tris pH 7.0, 10 mM EDTA, 0.01 %(v/v) Brij-35, and 0.1 % (v/v) CR-3.

Peptide sequences used with HCV NS3.
PEPTIDE SEQUENCE
NS4A Peptide NH2-KKGSVVIVGRIVLSGKPAIIPKK-COOH [SEQ ID NO:4]

NS5AB NH2-EDVV-(alpha)Abu-CSMSY-COOH [SEQ ID NO:2]
NS5AB-EDANS NH2-EDVV-(alpha)Abu-CSMSYT-Asp(EDANS)-KKK-COOH
[SEQ ID NO:5]

FITC-NS5AB-1 FITC-Ahx-EDVV-(alpha)Abu-CSMSYTKK-NH2 [SEQ ID NO:6]
5. Determination of Km and Vmax [00447] To determine the kinetic parameters Km and Vmax, the HCV NS3 protease domain or HCV NS3=4A was reacted with peptide substrate under the assay conditions described above. Peptide substrate concentration was varied between 3 M and 200 M, with less than 20 percent conversion at all substrate concentrations. The ratio of the product peak area (as determined by reverse phase HPLC) to the reaction time yielded a rate of enzyme catalyzed hydrolysis. These rate vs. substrate concentration data points were fit to the Michaelis-Menten equation using non-linear regression. The value of k'a, was determined from Vmax using the nominal protease concentration and a fully cleaved substrate peptide as an instrument calibration standard.
Kinetic parameters for peptide substrates with HCV NS3 or NS3 protease domain.
ENZYME SUBSTRATE Km ( M) k~at/Km (M- sec ) NS3 Protease Domain NS5AB 25 3.0 x 104 NS3=4A NS5AB 30 7.9 x 103 NS3=4A. NS5AB-EDANS 56 1.4 x 103 NS3=4A FITC-NS5AB-1 15 1.2 x 103 6. Determination of Compound Potency [00448] To evaluate apparent Ki values, all components except the test compound and substrate were pre-incubated for 5-10 minutes at room temperature. Then, test compound, dissolved in DMSO, was added to the mixture and incubated for either 15 minutes or 60 minutes at 30 C. Neat DMSO was included as a no inhibitor control. The cleavage reaction was initiated by the addition of peptide substrate at a concentration either equal to Km or equal to one-half times Km, and allowed to proceed at 30 C for twenty minutes. At the end of the reaction the mixture was quenched, and the extent of reaction was determined as described above. Eleven concentrations of compound were used to titrate enzyme activity for inhibition. Activity vs. inhibitor concentration data points were fit to the Morrison equation describing competitive tight-binding enzyme inhibition using non-linear regression (Sculley MJ and Morrison JF. Biochim. Biophys. Acta. 1986, 874, 44-53).
[00449] The tested compounds of formula I generally exhibited Ki values from about 0.100 to about 5 M. In some embodiments, the compounds of formula I exhibited Ki values from about 0.170 to about 3.7 M. In some other embodiments, the compounds of formula I
exhibited Ki values from about 0.150 to about 3.50 pM. In still some other embodiments, the compounds of formula I exhibited Ki values from 0.140 to about 3.200 M.
[00450] Examples of activities of the compounds of formulae (I, Ia, and Ib) on inhibiting :serine protease receptors are shown below in Table 3. For compound activities for serine protease measured using the HCV Enzyme Assays, serine protease activity is illustrated with "+++" if activity was measured to be less than 0.41 M, "++" if activity was measured to be from 0.41 M to 0.7 M, "+" if activity was measured to be greater than 0.7 M, and "-" if no data was available. It should be noted that 0% efficacy is the minimum response obtained with the DMSO only control. The Enzyme Assay 1 refers to the HCV NS3 Protease Domain Peptide Cleavage Assay and Enzyme Assay 2 refers to the HCV NS3 Peptide Cleavage Assay.
Table 3: HCV enzymatic assay activities and efficacies of exemplary compounds in accordance to Formulae I.
Cmpd. No. Activity Assay No. I Assay No. 2 1 ++

2 +
3 +++
4 +++
+

6 ++

7 +++

B. HCV Cell Assays [00451] Huh-7 cells were propagated in Dulbecco's modified Eagle's medium (DMEM, JRH
Biosciences, Lenexa, Kansas) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, and nonessential amino acids (JRH). The cells were transfected with an in vitro transcribed HCV replicon RNA identical to replicon I377neo/NS3-3'/wt as described by Lohmann et al. (1999). Stable cell clones were selected and maintained in the presence of 250 gg/mL G418 (Invitrogen, Carlsbad, California). One of the clones, 24-2, was used in the subsequent HCV replicon assays. The replicon cells were propagated in DMEM supplemented with 10% FBS, 2 mM L-glutamine, nonessential amino acids, and 250 g/mL G418. The cells were split twice per week in fresh media upon reaching confluence.
There are approximately 200 - 300 copies of HCV RNA per replicon cell.
[00452] HCV replicon RNA from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont California) as per the manufacturer's instructions.
Briefly, compound-treated replicon cells were lysed and immobilized on to capture plates using HCV
specific oligonucleotides over night and the relative amounts 6f captured RNA
was measured using oligonucleotide probe sets as per the manufacturer's instructions.
1. 2-Day HCV Replicon IC50 Assay [00453] On the day prior to the assay, 104 replicon cells were plated per well of a 96-well plate and allowed to attach and grow overnight in DMEM (Invitrogen, Carlsbad, California) supplemented with 10 % heat-inactivated FBS (JRH Biosciences, Lenexa, Kansas), 2 mM L-glutamine (Invitrogen), nonessential amino acids (Invitrogen) and 250 g/ml (Invitrogen). Compounds were serially diluted in DMEM plus 2 % FBS and 0.5%
DMSO
(Sigma Chemical Co., St. Louis, MO) without G418. HCV replicon RNA from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont California) as per the manufacturer's instructions. Briefly, compound-treated replicon cells were lysed and immobilized on to capture plates using HCV specific oligonucleotides overnight and the relative amounts of captured RNA was measured using oligonucleotide probe sets as per the manufacturer's instructions. Unless indicated otherwise, each data point represents the average of three replicates. The IC50 is the concentration of the compound at which the HCV

replicon RNA level in cells is reduced by 50 % as compared to the untreated replicon cell controls. To monitor the effect of compounds on cell proliferation or cell viability, replicon cells were treated with serially diluted compounds for 48 h,.after which cell viability was determined using a Ce1lTiter Glo assay (Promega, Madison, Wisconsin). Each CC50 is derived from three replicates and is the concentration of the compound at which the number of viable cells is reduced by 50 % as compared to untreated cell controls. The IC50 and CC50 was determined using 4 parameter curve fitting in the SoftMax Pro program (Molecular Devices, Sunnyvale, California).
2. 5-Day HCV Replicon IC99 Assay [00454] On the day prior to the assay, HCV replicon cells were plated at a low density of 2500 cells per well in a 96-well plate so the cells would not reach confluence during 5 days in culture. Compounds were serially diluted in DMEM containing 10 % FBS and 0.5 %
DMSO
in the absence of G418. Fresh media and compounds were added to the cells on day I and day 3. After the cells were treated with antiviral compounds for 5 days, HCV
replicon RNA
from cells was measured using the Quantigene Discover XL kit (Panomics Inc., Fremont California) as per the manufacturer's instructions. Briefly, compound-treated replicon cells were lysed and immobilized onto to capture plates using HCV specific oligonucleotides overnight and the relative amounts of captured replicon RNA was measured using oligonucleotide probe sets (Panomics) as per manufacturer's instructions. Each data point represents the average of two replicates. The IC99 is the concentration of the compound at which the HCV replicon RNA level in cells is reduced by 2 logs as compared to the untreated cell controls. To monitor the effect of compounds on cell proliferation or cell viability, replicon cells were treated with serially diluted compounds for 5 days, after which cell viability was determined using a CellTiter Glo assay (Promega, Madison, Wisconsin). Each CC50 is derived from two replicates and is the concentration of the compound at which the number of viable cells is reduced by 50% as compared to untreated cell controls. The IC99 and CC50 were determined by 4 parameter curve fitting method using the Prism software (GraphPad Software Inc., San Diego, California) and Excel program (Microsoft Corporation, Redmond, Washington).
[00455] Using the assays above, compounds of the present invention are determined to be useful serine protease inhibitors.
OTHER EMBODIMENTS
[00456] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein R1 is -Z A R8, wherein each Z A is independently a bond or an optionally substituted branched or straight C1-12 aliphatic chain wherein up to three carbon units of Z A are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR A-, -C(O)NR A NR A-, -C(O)O-, -NR A C(O)O-, -O-, -NR A C(O)NR A-, -NR A NR A-, -S-, -SO-, -SO2-, -NR A-, -SO2NR A-, or -NR A SO2NR A- provided that -O-, -NR A NR A-, -NR A C(O)NR A-, or -NR A
SO2NR A- is not directly bound to the nitrogen ring atom of formula I, Each R8 is independently R A, halo, -OH, -CN, -NO2, -NH2, or -OCF3, Each R A is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;
R2 and R3 are -Z B R9, wherein each Z B is independently a bond or an optionally substituted branched or straight C1-6 aliphatic chain wherein up to three carbon units of Z B are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR B-, -C(O)NR B
NR B-, -C(O)O-, -NR B C(O)O-, -NR B C(O)NR B-, -NR B NR B-, -S-, -SO-, -SO2-, -NR B-, -SO2NR B-, or -NR B SO2NR B-, Each R9 is independently RB, halo, -OH, -CN, -NO2, -NH2, or -OCF3, Each R B is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl, Or, R2 and R3, together form an oxo group;
A is -O-, or -CR6R7-, wherein each R6 and R7 is -Z C R10, wherein each Z C is independently a bond or an optionally substituted branched or straight C1-6 aliphatic chain wherein up to three carbon units of Z C are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR C-, -C(O)NR C NR C-, -C(O)O-, -NR C C(O)O-, -NR C C(O)NR C-, -NR C NR C-, -S-, -SO-, -SO2-, -NR C-, -SO2NR C-, or -NR C SO2NR C-, Each R10 is independently R C, halo, -OH, -CN, -NO2, -NH2, or -OCF3, Each R C is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;
R4 is -Z D R11, wherein each Z D is independently a bond or an optionally substituted branched or straight C1-12 aliphatic chain wherein up to three carbon units of Z D are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR D-, -C(O)NR D NR D-, -C(O)O-, -NR D C(O)O-, -O-, -NR D C(O)NR D-, -NR D NR D-, -S-, -SO-, -SO2-, -NR D -, -SO2NR D-, or -NR D SO2NR D-, provided that -SO-, -SO2-, or -SO2NR D- is not directly bound to the carbonyl adjacent to R4, Each R11 is independently R D, halo, -OH, -CN, -NO2, -NH2, or -OCF3, Each R D is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and R5 is -Z E R12, wherein each Z E is independently a bond or an optionally substituted branched or straight C1-12 aliphatic chain wherein up to three carbon units of Z E are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR E-, -C(O)NR E NR E-, -C(O)O-, -NR E C(O)O-, -O-, -NR E C(O)NR E-, -NR E NR E-, -S-, -SO-, -SO2-, -NR E-, -SO2NR E-, or -NR E SO2NR E-, Each R12 is independently R E, halo, -OH, -CN, -NO2, -NH2, or -OCF3, and Each R E is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;
Or, R4 and R5 together with the atoms to which they are attached form an optionally substituted heterocycloaliphatic ring.

2. The compound of claim 1, wherein R1 is an optionally substituted aliphatic.

3. The compound of any of claims 1-2, wherein R1 is an alkyl, alkenyl, or alkynyl, each of which is optionally substituted with 1-4 substituents independently selected from halo, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic.

4. The compound of any of claims 1-3, wherein R1 is an alkyl optionally substituted with 1-4 substituents independently selected from halo, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic.

5. The compound of any of claims 1-4, wherein R1 is methyl, ethyl, propyl, or butyl, each of which is optionally substituted with 1-4 substituents independently selected from halo, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic.

6. The compound of any of claims 1-5, wherein R1 is an unsubstituted alkyl.

7. The compound of any of claims 1-6, wherein R1 is an optionally substituted aralkyl.

8. The compound of any of claims 1-7, wherein R1 is an aryl-methyl, aryl-ethyl, or aryl-propyl, each of which is optionally substituted.

9. The compound of any of claims 1-8, wherein R1 is phenylmethyl, phenylethyl, or phenylpropyl, each of which is optionally substituted with 1-3 substituents independently selected from halo, aryl, hydroxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, and heteroaryl.

10. The compound of any of claims 1-9, wherein R1 is an optionally substituted aryl.

11. The compound of any of claims 1-10, wherein R1 is a monocyclic or bicyclic aryl, each of which is optionally substituted.

12. The compound of any of claims 1-11, wherein R1 is phenyl optionally substituted with 1-3 substituents selected from halo, hydroxy, aliphatic, aryl, heteroaryl, cycloaliphatic, and heterocycloaliphatic.

13. The compound of any of claims 1-12, wherein R2 and R3, together form an oxo group.

14. The compound of any of claims 1-13, wherein A is -O- or -CH2-.

15. The compound of any of claims 1-14, wherein each R4 is -Z1-V1-Z2-V2-Z3-V3 each of V1, V2, and V3 is independently a bond, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when V1, V2, V3 is the terminal group of R4; each of Z1, Z2, and Z3 is independently a bond, -C(O)-, -C(O)C(O)-, -C(S)-, -C(O)N(Q6)-, -N(Q6)C(O)-, -C(O)C(O)N(Q6)-, -O-, , SO-, -SO2-5 -N(Q6)SO2-, -N(Q6)C(O)N(Q6)-, -N(Q6)C(S)N(Q6)-, -N(Q6)-, -N(Q6)SO2-, -SO2N(Q6)-, -C(O)N(Q6)SO2-, -SO2N(Q6)C(O)-, or hydrogen when Z1, Z2, or Z3 is the terminal group of R2;
and each Q6 is independently hydrogen, or an optionally substituted aliphatic.

16. The compound of any of claims 1-15, wherein R4 is an optionally substituted (aliphatic)amino, optionally substituted (cycloaliphatic)amino, an optionally substituted alkoxy, or hydroxy.

17. The compound of any of claims 1-16, wherein R4 is an optionally substituted (aliphatic)amino wherein the aliphatic portion of R4 is -Z2-V2-Z3-V3 or -Z3-V3 wherein each of Z2 and Z3 is independently a bond, -C(O)-, -N(Q5)-, -CH(OH)-, -C(O)N(Q6)-, or -C(O)C(O)N(Q6)-; V2 is independently a bond, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic; and V3 is hydrogen, an optionally substituted aliphatic, or an optionally substituted cycloaliphatic.

18. The compound of any of claims 1-17, wherein Z2 is -CH(OH)-, V2 is a bond, and Z3 is -C(O)N(Q6)- such that R4 is -N(Q6)-CH(OH)-C(O)-N(V3)(Q6).

19. The compound of any of claims 1-18, wherein R4 is an alkoxy optionally substituted with 1-3 of halo, hydroxy, aliphatic, cycloaliphatic, or heterocycloaliphatic.

20. The compound of any of claims 1-19, wherein R4 is amino.

21. The compound of any of claims 1-20, wherein R4 is a (cycloaliphatic(carbonyl(carbonyl(alkyl))))amino, (amino(carbonyl(carbonyl(aliphatic))))amino, (aliphatic(carbonyl(carbonyl(aliphatic))))amino, or (aryl(amino(carbonyl(carbonyl(aliphatic)))))amino, each of which is optionally substituted.

22. The compound of any of claims 1-21, wherein R4 is -NR4 ZR'4Z, -SR4Y, or -NR4Y-CR4X R'4x-L1 -NR4Z-R4W, wherein R4Y is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;
each R4W is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; each R4X and R'4X is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R4X and R'4X together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring;
each L1 is -CH2-, -C(O)-, -CF2-, -C(O)C(O)-, -C(O)O-, -S(O)-, or -SO2-; each R4Z or R'4Z is hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4z and R'4Z together with the nitrogen to which they are both attached may form an optionally substituted 3 to 7 membered heterocycloaliphatic ring.

23. The compound of any of claims 1-22, wherein each R4X and R'4X is independently hydrogen, or optionally substituted aliphatic, optionally substituted cycloaliphatic, or optionally substituted (cycloaliphatic)aliphatic.

24. The compound of any of claims 1-23, wherein L1 is -C(O)C(O)- or -SO2-.

25. The compound of any of claims 1-24, wherein each R4W is hydrogen or optionally substituted cycloaliphatic.

26. The compound of any of claims 1-25, wherein R4 is -NH-CHR4X-C(O)-C(O)-N(R4Z)R4W.

27. The compound of any of claims 1-26, wherein R4 is -NH-CHR4X-CH(OH)-C(O)-N(R4Z)R4W.

28. The compound of any of claims 1-27, wherein R4 is -NH-CHR4X-C(O)-C(O)-NH-cyclopropyl.

29. The compound of any of claims 1-28, wherein R4 is:

, wherein R4X is ; and R4W is , or hydrogen.

30. The compound of any of claims 1-29, wherein R4 is or wherein each R56 is independently optionally substituted C1-6 aliphatic;

optionally substituted aryl, optionally substituted heteraryl, optionally substituted cycloaliphatic, or optionally substituted heterocycloaliphatic; each R57 is independently optionally substituted aliphatic, optionally substituted aryl, optionally substituted aliphatic, optionally substituted heteroaryl, optionally substituted aliphatic, optionally substituted cycloaliphatic or optionally substituted amino; and m is 1 or 2; and each R4X
and R'4X is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4X and R'4X together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.

31. The.compound of any of claims 1-30, wherein R4 is wherein R58 and R59 are each independently selected from optionally substituted aliphatic, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted (cycloaliphatic)oxy, optionally substituted (heterocycloaliphatic)oxy optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloaliphatic or optionally substituted amino; and each R4X and R'4X is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; or R4X and R'4X together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring.

32. The compound of any of claims 1-31, wherein R4 is one selected from the group consisting of , where X200 is -OX202 OR -X202, and X202 is aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, or heteroaryl.

33. The compound of claim 1, wherein R4 is

34. The compound of any of claims 1-33, wherein R4 is

35. The compound of any of claims 1-34, wherein R5 is -Q4-W4-Q3-W3-Q2-W2-Q1;
wherein each of W2, W3, and W4 is independently a bond, -C(O)-, -C(S)-, -C(O)N(Q5)-, -C(O)O-, -0-, -N(Q5)C(O)N(Q5)-, -SO2-, -N(Q5)SO2-, -S-, -N(Q5)-, -SO-, -OC(O)-, -N(Q5)C(O)O-, or -SO2N(Q5)-; each of Q1, Q2, Q3 and Q4 is independently a bond, an optionally substituted C1-4 aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, an optionally substituted heteroaryl, or a hydrogen when Q1, Q2, Q3, or Q4 is the terminal group of R5;
and each Q5 is independently hydrogen or an optionally substituted aliphatic.

36. The compound of any of claims 1-35, wherein Q4 is a bond.

37. The compound of any of claims 1-36, wherein R5 is an optionally substituted acyl group.

38. The compound of any of claims 1-37, wherein R5 is (amino)alkylcarbonyl, (halo)alkylcarbonyl, (ar.yl)alkylcarbonyl, (cycloaliphatic)alkylcarbonyl, or (heterocycloaliphatic)alkylcarbonyl, (heterocycloalkyl(oxy(carbonyl(amino))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino)))))alkylcarbonyl, (bicycloaryl(sulfonyl(amino)))alkylcarbonyl, (aryl(alkoxy(carbonyl(amino))))alkylcarbonyl, (alkyl(carbonyl(amino)))alkylcarbonyl, (alkenyl(alkoxy(carbonyl(amino))))alkylcarbonyl, (cycloaliphatic(alkyl(amino(carbonyl(amino)))))alkylcarbonyl, (heteroaryl(carbonyl(amino(alkyl(carbonyl(amino))))))alkylcarbonyl, (alkyl(amino(carbonyl(amino))))alkylcarbonyl, or (bicycloaryl(amino(carbonyl(amino))))alkylcarbonyl, each of which is optionally substituted.

39. The compound of any of claims 1-38, wherein R5 is a heteroarylcarbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic(oxy(amido(alkyl))))carbonyl, an (aryl(sulfonyl(amino(alkyl))))carbonyl, an (aralkyl(oxy(amido(alkyl))))carbonyl, an (aliphatic(oxy(amido(alkyl))))carbonyl, a (cycloaliphatic(alkyl(amido(alkyl))))carbonyl, a (heterocycloaliphatic)carbonyl, or a (heteroaryl(amido(alkyl(amido(alkyl))))carbonyl, each of which is optionally substituted with 1-4 of halo, aliphatic, cycloaliphatic, acyl, alkoxy, or combinations thereof.

40. The compound of any of claims 1-39, wherein R5 is an optionally substituted carboxy group.

41. The compound of any of claims 1-40, wherein R5 is an (aliphatic(oxy))carbonyl, a (heteroaralkyl(oxy))carbonyl, (heterocycloaliphatic(oxy)carbonyl, (aralkyl(oxy))carbonyl, each of which is optionally substituted with 1-3 of halo, alkoxy, aliphatic, cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, or combinations thereof.

42. The compound of any of claims 1-41, wherein R5 is an amido.

43. The compound of any of claims 1-42, wherein R5 is (alkoxy(aryl(alkyl)))aminocarbonyl, (alkyl)aminocarbonyl, or (aryl(alkoxy(carbonyl(alkyl(amino(carbonyl(alkyl)))))))aminocarbonyl, each of which is optionally substituted.

44. The compound of any of claims 1-43, wherein R5 is an alkylsulfonyl, aminosulfonyl, arylsulfonyl, heteroarylsulfonyl, cycloaliphaticsulfonyl, or heterocycloaliphaticsulfonyl, each of which is optionally substituted.

45. The compound of any of claims 1-44, wherein R5 is an optionally substituted alkylsulfonyl.

46. The compound of any of claims 1-45, wherein R5 is (aryl)alkylsulfonyl, or (alkyl(amino))alkylsulfonyl, each of which is optionally substituted.

47. The compound of any of claims 1-46, wherein R5 is wherein T is a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)- or -SO2-; each R
is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R13 and R'13 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R14 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R13 and R14, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic; or R13 and R'13, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic.

48. The compound of any of claims 1-47, wherein R in the substituent in QI, QII, QIII, QIV, QV, or QVI is

49. The compound of any of claims 1-48, wherein R5 is QVI and R is

50. The compound of any of claims 1-49, wherein R in the substituent in QI, QII, QIII, QIV, QV, or QVI is wherein each R15 and R'15 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic, or R15 and R'15 together with the atom to which they are both bound form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic; and each K is independently a bond, C1-12 aliphatic, -O-, -S-, -S(O)2-, -NR16-, -C(O)-, or -C(O)NR16-, wherein R16 is hydrogen or an optionally substituted C1-12 aliphatic; and n is 1-3. For clarity, when more than one R15 is present in QI, QII, QIII, QIV, QV, or QVI, each R15 can be the same or different.

51. The compound of any of claims 1-50, wherein R15 is [C3-10 cycloalkyl or cycloalkenyl]-C1-12 aliphatic, (3 to 10 membered)-heterocycloaliphatic, (3 to 10 membered)-heterocycloaliphatic-C1-12 aliphatic-, (5 to 10 membered)-heteroaryl, or (5 to 10 membered)-heteroaryl-C1-12 aliphatic-.

52. The compound of any of claims 1-51, wherein R in the substituent in QI, QII, QIII, QIV, QV, or QVI is

53. The compound of any of claims 1-52, wherein R in the substituent in QI, QII, QIII, QIV, QV, or QVI is wherein each Z is independently -O-, -S-, -NR50-, or -C(R50)2-, is independently a single bond or a double bond, and each R50 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic; and n is 1 or 2.

54. The compound of any of claims 1-53, wherein R5 is wherein wherein T is a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)-or -SO2-; each R is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; each R13 and R'13 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R14 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, or R13 and R14, bound on adjacent atoms, taken together with the atoms to which they are attached form a 5 to 7 membered, optionally substituted monocyclic heterocycloaliphatic, or a 6 to 12 membered, optionally substituted bicyclic heterocycloaliphatic, in which each heterocycloaliphatic ring; or R13 and R'13, taken together with the atoms to which they are attached form an optionally substituted cycloaliphatic or an optionally substituted heterocycloaliphatic; each R15 and R'15 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic; or R15 and R'15 together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring; and each R12 is independently hydrogen or a protecting group.

55. The compound of any of claims 1-54, wherein R15 and R'15 together with the atom to which they are attached form a 3 to 7 membered ring.

56. The compound of any of claims 1-55, wherein R15 and R'15 together with the atom to which they are attached form

57. The compound of claim 21, wherein each of R13 and R15 is independently hydrogen,

58. The compound of any of claims 1-57, wherein R13 and R15 together with the atoms to which they are attached may form an optionally substituted 5 to 7 membered monocyclic heterocycloaliphatic or an optionally substituted 6 to 12 membered bicyclic heterocycloaliphatic.

59. The compound of any of claims 1-58, wherein R5 is: wherein T is -C(O)-, and R is

60. The compound of any of claims 1-59, wherein R5 is: , wherein R13 is T is -C(O)-, and R is

61. The compound of any of claims 1-61, wherein R5 is one selected from the group consisting of where X99 = OR, OC(NH)R, or ; and each R is independently hydrogen, amino, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

62. The compound of any of claims 1-61, wherein R4 and R5, together with the atoms to which they are attached, form an optionally substituted heterocycloaliphatic ring that has 1-3 heteroatoms selected from N, O, and S.

63. The compound of any of claims 1-62 having the structure wherein each R4w is independently or hydrogen; each T
is independently a bond, -C(O)-, -OC(O)-, -NHC(O)-, -S(O)2N(H)-, -C(O)C(O)- or -SO2-;
each R is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl; and each R14 is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl.

64. The compound of any of claims 1-63 having the structure

65. A compound of formula II:

or a pharmaceutically acceptable salt thereof, wherein R1a is an optionally substituted aryl or an optionally substituted heteroaryl;
R4Y is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;
R4w is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic;
R4X and R'4X is independently hydrogen, an optionally substituted aliphatic, an optionally substituted heteroaryl, an optionally substituted phenyl, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, Or R4X and R'4X together with the atom to which they are both attached form an optionally substituted 3 to 7 membered cycloaliphatic or heterocycloaliphatic ring;
R4Z is hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl, Or R4Z and R4W together with the nitrogen to which they are both attached may form an optionally substituted 3 to 7 membered heterocycloaliphatic ring;
R14 is independently hydrogen, optionally substituted aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycloaliphatic, or optionally substituted cycloaliphatic;
R5b is -Z F R21, wherein Z F is -CH2-, -NH-, -CH(R22)-, or -O-, R21 is optionally substituted 6-7 membered cycloaliphatic or optionally substituted tert-butyl, and R22 is optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocycloaliphatic, optionally substituted aryl, or optionally substituted heteroaryl; and A is -O-, or -CR6R7-, wherein each R6 and R7 is -Z C R10, wherein each Z C is independently a bond or an optionally substituted branched or straight C1-6 aliphatic chain wherein up to three carbon units of Z C are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR C-, -C(O)NR C NR C-, -C(O)O-, -NR C C(O)O-, -NR C C(O)NR C-, -NR C NR C-, -S-, -SO-, -SO2-, -NR C-, -SO2NR C-, or -NR C SO2NR C-;
Each R10 is independently R C, halo, -OH, -CN, -NO2, -NH2, or -OCF3; and Each R C is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

66. A compound of formula III
or a pharmaceutically acceptable salt thereof, wherein R5e is: R4e is:

R'4e is:

or hydrogen;
R1e is optionally substituted aryl or optionally substituted heteroaryl; and A is -O-, or -CR6R7-, wherein each R6 and R7 is -Z C R10, wherein each Z C is independently a bond or an optionally substituted branched or straight C1-6 aliphatic chain wherein up to three carbon units of Z C are optionally and independently replaced by -C(O)-, -C(S)-, -C(O)NR C-, -C(O)NR C NR C-, -C(O)O-, -NR C C(O)O-, -NR C C(O)NR C-, -NR C NR C-, -S-, -SO-, -SO2-, -NR C-, -SO2NR C-, or -NR C SO2NR C-, Each R10 is independently R C, halo, -OH, -CN, -NO2, -NH2, or -OCF3, and Each R C is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl.

67. A compound having the structure of compound selected from the group of compound numbers 1 through 9.

68. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof in an amount effective to inhibit a serine protease;
and an acceptable carrier, adjuvant or vehicle.

69. A pharmaceutical composition comprising a compound according to claim 67 or a pharmaceutically acceptable salt thereof in an amount effective to inhibit a serine protease;
and an acceptable carrier, adjuvant or vehicle.

70. The composition according to claim 68, wherein said composition comprises an additional agent selected from an immunomodulatory agent; an antiviral agent;
a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; and a cytochrome P-450 inhibitor; or combinations thereof.

71. The composition according to claim 69, wherein said composition comprises an additional agent selected from an immunomodulatory agent; an antiviral agent;
a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; and a cytochrome P-450 inhibitor; or combinations thereof.

72. The composition according to claim 70, wherein said immunomodulatory agent is .alpha.-, .beta.-, or .gamma.-interferon or thymosin; said antiviral agent is ribavirin, amantadine, or telbivudine;
or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.

73. The composition according to claim 71, wherein said immunomodulatory agent is .alpha.-, .beta.-, or .gamma.-interferon or thymosin; said antiviral agent is ribavirin, amantadine, or telbivudine;
or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.

74. The composition according to claim 70, wherein said cytochrome P-450 inhibitor is ritonavir.

75. The composition according to claim 71, wherein said cytochrome P-450 inhibitor is ritonavir.

76. A method of inhibiting the activity of a serine protease comprising the step of contacting said serine protease with a compound according to claim 1.

77. A method of inhibiting the activity of a serine protease comprising the step of contacting said serine protease with a compound according to claim 67.

78. The method according to claim 76, wherein said serine protease is an HCV

protease.

79. The method according to claim 77, wherein said serine protease is an HCV

protease.

80. A method of treating an HCV infection in a patient comprising the step of administering to said patient a compound according to claim 1.

81. A method of treating an HCV infection in a patient comprising the step of administering to said patient a compound according to claim 67.

82. The method according to claim 80, further comprising administering to said patient an additional agent selected from an immunomodulatory agent; an antiviral agent;
a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; or combinations thereof; wherein said additional agent is administered to said patient in the same dosage form as the serine protease inhibitor or as a separate dosage form.

83. The method according to claim 81, further comprising administering to said patient an additional agent selected from an immunomodulatory agent; an antiviral agent;
a second inhibitor of HCV protease; an inhibitor of another target in the HCV life cycle; or combinations thereof; wherein said additional agent is administered to said patient in the same dosage form as the serine protease inhibitor or as a separate dosage form.

84. The method according to claim 82, wherein said immunomodulatory agent is .alpha.-, .beta.-, or .gamma.-interferon or thymosin; said antiviral agent is ribavarin or amantadine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.

85. The method according to claim 83, wherein said immunomodulatory agent is .alpha.-, .beta.-, or .gamma.-interferon or thymosin; said antiviral agent is ribavarin or amantadine; or said inhibitor of another target in the HCV life cycle is an inhibitor of HCV helicase, polymerase, or metalloprotease.

86. A method of eliminating or reducing HCV contamination of a biological sample or medical or laboratory equipment, comprising the step of contacting said biological sample or medical or laboratory equipment with a compound according to claim 1.

87. A method of eliminating or reducing HCV contamination of a biological sample or medical or laboratory equipment, comprising the step of contacting said biological sample or medical or laboratory equipment with a compound according to claim 67.